Stress-responsive activator of p300 (strap) protein

ABSTRACT

The invention provides a protein which is a stress-responsive activator of the p300 protein, and nucleic acid sequences encoding the protein. The protein performs a key role in facilitating stress-responsive protein-protein interactions within the p300 co-activator complex. The STRAP protein facilitates the interaction of other proteins in the p300 complex, and is thus a target for assays for modulators of the complex.

FIELD OF INVENTION

[0001] This invention relates to the isolation and characterisation of anovel polypeptide (Stress Responsive Activator of p53: STRAP) which isshown to interact with p300 and the p300 co-factor JMY in the p300co-activator complex. This complex is involved in the regulation of thetranscription of p53 target genes.

BACKGROUND OF INVENTION

[0002] The p53 protein is a stress-responsive transcription factor whichis induced by a variety of stimuli that act through mechanisms thatalter p53 half-life (Ko and Prives, 1997; Levine, 1997). It is encodedby a tumour suppressor gene that is frequently mutated in human cancercells (Greenblat, 1994). In normal cells p53 exerts its function throughthe targeted sequence-specific activation of a number of differentp53-responsive genes. These genes include waf1, bax, mdm2 and gadd45which encode proteins that give rise to the physiological consequencesof p53 activation, namely apoptosis or cell cycle arrest (Ko and Prives,1997). p53 alleles isolated from tumour cells frequently harbourmutations that disrupt p53 DNA binding activity (Cho et al., 1994),underscoring the importance of transcription-related functions inmediating the effects of p53 tumour suppression.

[0003] The pathways through which p53 activity is regulated have beensubject to intense study. The transcriptional activation domain of p53is targeted by the MDM2 oncoprotein, which thereafter prevents p53 fromactivating transcription by hindering the interaction of p53 with thetranscription apparatus (Oliner et al., 1993; Lin et al., 1994). In thisrespect, MDM2 can override the physiological effects of p53 response (Wuet al., 1993), and it is consistent with this idea that mdm2 is oftenseen to be aberrantly expressed in human tumour cells (Piette et al.,1997). A further consequence of the interaction between MDM2 and p53 isa down-regulation in the level of p53 protein, which is mediated in partthrough a ubiquitin-dependent pathway and requires the MDM2 E3 ligase(Haupt et al., 1997; Honda et al., 1997; Kubbutat et al., 1997).

[0004] Because p53 inactivation is associated with many human cancers,research has been directed at restoring p53 function, in order toprovide a therapy for these cancers. P53 may also make normal cellssensitive to stress and recently, research has also been directed at thetemporary inhibition of p53 (Komarova E. and Gudkov A. (1998) Seminarsin Cancer Biology 8(5) 389-400). This inhibition may be useful inameliorating the p53 induced side effects of cancer therapies such asradio-and chemo-therapy, which include hair loss and damage to thelymphoid and haematopoietic systems and the intestinal epithelia.

[0005] Adverse effects associated with the activation of P53 have alsobeen described in other conditions including injury associated cellularstress (e.g. burns), diseases associated with fever, local hypoxiaconditions associated with a deficient blood supply (e.g. stroke andischaemia) and cell aging (e.g. fibroblast senescence). Suppression ofp53 activity may therefore be useful in therapies related to theseconditions.

[0006] A considerable body of evidence supports a role for the p300/CBPfamily of co-activators in p53-dependent transcription (Shikama et al.,1997). For example, p300/CBP proteins physically interact with the p53activation domain, and dominant-negative derivatives of p300/CBPproteins can block p53 activity (Avantaggiati et al., 1997; Gu et al.,1997; Lill et al., 1997; Lee et al., 1998). Moreover, phosphorylation ofthe p53 activation domain during the stress response is believed toantagonise the interaction with MDM2, and possibly stabilise theinteraction with p300/CBP proteins (Sheih et al., 1997; Giaccia andKastan, 1998; Chehab et al., 2000; Shieh et al., 2000). However,p300/CBP proteins function as integral components of largermulticomponent co-activator complexes, and a variety of co-factors thatmake up p300/CBP complexes have been identified (Shikama et al., 1997;Shiltz and Nakatani, 2000). Of considerable interest is the JMYco-factor, which is an integral component of the p300 co-activatorcomplex that augments the transcriptional activity of p53, and enhancesthe level of the p53 response (Shikama et al., 1999). No othercomponents of the p300 complex transcription have been characterisedwhich are involved in regulating p53.

SUMMARY OF INVENTION

[0007] The present inventors have isolated and characterised a newcomponent of the p300 co-activator complex, which has been termed STRAP.

[0008] STRAP is composed almost entirely of a tandem array oftetratricopeptide (TPR) motifs (Lamb et al 1995) and can interact withdistinct components of the p300 co-activator complex, such as p300 andthe p300 co-factor JMY. STRAP augments the association between p300 andJMY and can induce the p53 response, providing indication that STRAPplays a role in regulating the assembly of the p300 co-activator-complexunder stress conditions.

[0009] STRAP also undergoes a stress-responsive protein accumulation.The ability of STRAP to promote the interaction of co-factors within thep300 complex, combined with its response under cellular stress, endowSTRAP with a critical role in regulating the p53 response. Takentogether, these results identify a new and hitherto unexpected level ofcontrol, mediated at the point of stress-responsive co-activatorcontrol, in the cellular response to stress.

BRIEF DESCRIPTION OF THE FIGURES

[0010]FIG. 1 shows the primary amino acid sequence of murine STRAP (440amino acid residues, FIG. 1A, SEQ ID NO:1), human STRAP (440 aminoacids, FIG. 1B, SEQ ID NO:2), and the alignment of the two sequences(FIG. 1C).

[0011]FIG. 2 shows the cDNA nucleic acid sequence (SEQ ID NO:3) encodingthe STRAP amino acid sequence shown in FIG. 1A (SEQ ID NO:1).

[0012]FIG. 3 shows a diagrammatic summary of the distribution of the sixTPR motifs in STRAP (each TPR motif is indicated as I to VI andhighlighted).

[0013]FIG. 4 shows an alignment of the sequence from the six TPR motifs(SEQ ID NOs:4-9) in STRAP. The eight consensus residues derived from thealignment are shown below. The residue number is indicated at each endof the TPR motif. The consensus TPR motif is taken from Blatch andLassle (1999).

[0014]FIG. 5 shows the results of a two-hybrid assay in mammalian cells:the indicated expression vectors, either pVP16-JMY (0.5 μg) or pG4-Straptogether with the control (0.5 μg), pVP16 (0.5 μg) or G4 (0.1 μg)vectors were introduced into U2OS cells as indicated, together with thereporter pG5-luc (0.5 μg) and internal control pCMV-β-gal (1 μg). Thedata represent the relative activity of luciferase to β-galactosidaseand are the average values derived from two or more independentexperiments.

[0015]FIG. 6 shows the results of a two-hybrid assay performed in U2OScells as described in a) using the indicated vectors, namely pVP16-Strap(0.5 μg) or pG4-p300 (0.5 μg). The data represent the average valuesderived from two or more independent experiments.

[0016]FIG. 7 shows a summary of the binding domains in Strap, JMY andp300. The binding domains in JMY for p300, and in p300 for JMY, aretaken from Shikama et al., 1999.

[0017]FIG. 8 shows the results of a two-hybrid assay performed in U2OScells with pG4-p300 (50 ng) and pVP-16-JMY (250 ng) in the presence ofincreasing amounts of pHA-Strap (0.5, 1.0, 3.0 and 5.0 μg), togetherwith the reporter pG5-luc (1 μg) and internal control pCMV-βgal (1 μg).The data represent the relative activity of luciferase toβ-galactosidase and are the average values derived from two or moredifferent readings.

[0018]FIG. 9 shows the indicated p53 reporter constructs, pWWP-luc,pBax-luc and pTG13 (1 μg) together with expression vectors for p53(0.025 μg) and Strap (2 μg and 5 μg) were transfected into SAOS2 cellsas indicated. The values shown represent the average of three separatereadings and are the relative level of luciferase to β-galactosidasederived from the internal control.

[0019]FIGS. 10 and 11 show the results of SAOS2 cells transfected withexpression vectors for wild-type p53 or p5322/23 (4 μg) in the presenceor absence of Strap (7 μg) together with pCMV CD20 (7 μg), as indicated.At 36 h after transfection, transfected cells were identified bystaining with anti-CD20 antibody, and DNA was stained with propidiumiodide, and the proportion of sub-G1, G1, S and G2/M phase cellsdetermined as described. The % change in the size of the sub-G1, G1, Sand G2/M population is shown. p53 caused about 37% of the transfectedcells to enter apoptosis, compared to 17% with the vector alone.

[0020]FIG. 12 shows the results of COS cells transfected with eitherpHA-Strap (5 μg), pCMV-JMY (5 μg) or pCMV-NAP2 (5 μg) and treated asdescribed either with or without etoposide (200 or 400 nM). The levelsof exogenous Strap, JMY and NAP, and endogenous p53, were determined byimmunoblotting with the relevant antibodies, quantitated byphosphoimaging and thereafter presented graphically. The level ofprotein detected in the non-treated cell was given an arbitrary value of1.0. The symbols indicate: • p53, □ Strap, ▪ JMY and ∘ NAP.

[0021]FIG. 13 shows a two-hybrid assay performed in U2OS cells withpG4-p300 (100 ng) and pVP16-JMY (1 μg) in the presence of pHA-Strap (2.5μg) and etoposide (200 nM) as indicated, and the reporter pG5-luc (1 μg)and internal control pCMV-βgal (1 μg). The data represent the relativeactivity of luciferase to β-galactosidase, and are the average valuesderived from two different readings.

[0022]FIG. 14 shows SAOS2 cells transfected with expression vectors forStrap or Strap8-123 (4 μg) and treated with etoposide (100 and 200 nM)as indicated. Exogenous p53 transcriptional activity was measured usingpBax-luc (1 μg), which was co-transfected together with expressionvectors for p53 (0.025 μg) and the internal control pCMV-βgal (1 μg).The values shown represent the average of three separate readings andare the relative level of luciferase.

[0023]FIG. 15 shows a possible model for role of Strap in the p53response. Strap is induced by stress, and activates p300 co-activatorfunction. As a result, the p53 response is triggered to cause apoptosisor G1 arrest. Stress activated phosphokinases (including ATM, ATR andchkl/2) act upon p53 and MDM2 to alter p53 stability and enhance theinteraction of p53 with the p300 co-activator complex. Strap is inducedby stress (? indicates that it is not known whether the samephosphokinases are involved) and augments the activity of the p300co-activator complex by facilitating co-factor interactions.

DETAILED DESCRIPTION OF INVENTION

[0024] According to one aspect of the present invention there isprovided an isolated nucleic acid molecule encoding a polypeptide whichincludes the amino acid sequence shown in FIG. 1A (SEQ ID NO:1) or 1B(SEQ ID NO:2). For convenience, the sequences are-referred to herein asthe sequence shown in FIG. 1, and reference to the sequence of FIG. 1 isintended to include both sequences unless specified explicitly to thecontrary.

[0025] The coding sequence may be that shown included in FIG. 2 (SEQ IDNO:3) or it may be a mutant, variant, derivative or allele of thesequence shown. A mutant, variant, derivative or allele may differ fromthe sequence shown by a change which is one or more of addition,insertion, deletion and substitution of one or more nucleotides of thesequence shown. Changes to a nucleotide sequence may result in an aminoacid change at the protein level, or not, as determined by the geneticcode.

[0026] Thus, nucleic acid according to the present invention may includea sequence different from the sequence shown in FIG. 2 (SEQ ID NO:3) yetencode a polypeptide with the same amino acid sequence. The amino acidsequence shown in FIG. 1 (SEQ ID NO:1 or SEQ ID NO-2) consists of 440residues.

[0027] Alternatively, the encoded polypeptide may comprise an amino acidsequence which differs by one or more amino acid residues from the aminoacid sequence shown in FIG. 1 (SEQ ID NO:1 or SEQ ID NO:2). Nucleic acidencoding a polypeptide which is an amino acid sequence mutant, variant,derivative or allele of the sequence shown in FIG. 1 (SEQ ID NO:1 or SEQID NO:2) is further provided by the present invention. Such polypeptidesare discussed below. Nucleic acid encoding such a polypeptide may showat the nucleotide sequence and/or encoded amino acid level greater thanabout 60% homology with the coding sequence shown in FIG. 2 (SEQ IDNO:3) and/or the amino acid sequence shown in FIG. 1 (SEQ ID NO:1 or SEQID NO:2), greater than about 70% homology, greater than about 80%homology, greater than about 90% homology or greater than about 95%homology.

[0028] For amino acid “homology”, this may be understood to besimilarity (according to the established principles of amino acidsimilarity, e.g. as determined using the algorithm GAP (GeneticsComputer Group, Madison, Wis.) or identity. GAP uses the Needleman andWunsch algorithm to align two complete sequences that maximizes thenumber of matches and minimizes the number of gaps. Generally, thedefault parameters are used, with a gap creation penalty=12 and gapextension penalty=4. Use of GAP may be preferred but other algorithmsmay be used, e.g. BLAST (which uses the method of Altschul et al. (1990)J. Mol. Biol. 215: 405-410), FASTA (which uses the method of Pearson andLipman (1988) PNAS USA 85: 2444-2448), or the Smith-Waterman algorithm(Smith and Waterman (1981) J. Mol. Biol. 147: 195-197), generallyemploying default parameters.

[0029] Another method for determining the best overall match between annucleotide or amino acid sequence of the present invention, or a portionthereof, and a query sequence is the use of the FASTDB computer programbased on the algorithm of Brutlag et al (Comp. App. Biosci., 6; 237-245(1990)). The program provides a global sequence alignment. The result ofsaid global sequence alignment is in percent identity. Suitableparameters used in a FASTDB search of a DNA sequence to calculatepercent identity are: Matrix=Unitary, k-tuple=4, Mismatch penalty=1,Joining Penalty=30, Randomization Group Length=0, Cutoff Score=1, GapPenalty=5, Gap Size Penalty=0.05, and Window Size=500 or query sequencelength in nucleotide bases, whichever is shorter. Suitable parameters tocalculate percent identity and similarity of an amino acid alignmentare: Matrix=PAM 150, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20,Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap SizePenalty=0.05, and Window Size=500 or query sequence length in nucleotidebases, whichever is shorter.

[0030] Use of either of the terms “homology” and “homologous” hereindoes not imply any necessary evolutionary relationship between comparedsequences, in keeping for example with standard use of terms such as“homologous recombination” which merely requires that two nucleotidesequences are sufficiently similar to recombine under the appropriateconditions. Further discussion of polypeptides according to the presentinvention, which may be encoded by nucleic acid according to the presentinvention, is found below.

[0031] The present invention extends to nucleic acid that hybridizeswith any one or more of the specific sequences disclosed herein understringent conditions. Such nucleic acid may include other animal, forexample fish (such as the Zebra fish), worm (such as C. elegans) andparticularly mammalian (e.g. rat or rabbit, sheep, goat, pig, or primateparticularly human) homologues of the STRAP gene. Such sequences may beobtained by making or obtaining cDNA libraries made from dividing cellsor tissues or genomic DNA libraries from other animal species, andprobing such libraries with probes comprising all or part of a nucleicacid of the invention under conditions of medium to high stringency.

[0032] Suitable conditions include, e.g. for detection of sequences thatare about 80-90% identical suitable conditions-include hybridizationovernight at 42° C. in 0.25M Na2HPO4, pH 7.2, 6.5% SDS, 10% dextransulfate and a final wash at 55° C. in 0.1×SSC, 0.1% SDS. For detectionof sequences that are greater than about 90% identical, suitableconditions include hybridization overnight at 65° C. in 0.25M Na2HPO4,pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 60° C. in0.1×SSC, 0.1% SDS.

[0033] A variant form of a nucleic acid molecule may contain one or moreinsertions, deletions, substitutions and/or additions of one or morenucleotides compared with the wild-type sequence (such as shown in FIG.2(SEQ ID NO:3)) which may or may not disrupt the gene function.Differences at the nucleic acid level are not necessarily reflected by adifference in the amino acid sequence of the encoded polypeptide.However, a mutation or other difference in a gene may result in aframe-shift or stop codon, which could seriously affect the nature ofthe polypeptide produced (if any), or a point mutation or grossmutational change to the encoded polypeptide, including insertion,deletion, substitution and/or addition of one or more amino acids orregions in the polypeptide. A mutation in a promoter sequence or otherregulatory region may prevent or reduce expression from the gene oraffect the processing or stability of the mRNA transcript. For instance,a sequence alteration may affect splicing of mRNA.

[0034] Generally, nucleic acid according to the present invention isprovided as an isolate, in isolated and/or purified form, or free orsubstantially free of material with which it is naturally associated,such as free or substantially free of nucleic acid flanking the gene inthe human genome, except possibly one or more regulatory sequence(s) forexpression. Nucleic acid may be wholly or partially synthetic and mayinclude genomic DNA, cDNA or RNA. The coding sequence shown herein is aDNA sequence. Where nucleic acid according to the invention includesRNA, reference to the sequence shown should be construed as encompassingreference to the RNA equivalent, with U substituted for T.

[0035] Nucleic acid may be provided as part of a replicable vector, andalso provided by the present invention are a vector including nucleicacid as set out above, particularly any expression vector from which theencoded polypeptide can be expressed under appropriate conditions, and ahost cell containing any such vector or nucleic acid. An expressionvector in this context is a nucleic acid molecule including nucleic acidencoding a polypeptide of interest and appropriate regulatory sequencesfor expression of the polypeptide, in an in vitro expression system,e.g. reticulocyte lysate, or in vivo, e.g. in eukaryotic cells such asCOS or CHO cells or in prokaryotic cells such as E. coli. This isdiscussed further below.

[0036] The nucleic acid sequence provided in accordance with the presentinvention is also useful in methods for identifying and/or obtainingnucleic acid of interest (and which may be according to the presentinvention) in a test sample, for example, homologues of the STRAPnucleotide sequence as described above.

[0037] A method of identifying and/or obtaining nucleic acid of interestmay include hybridisation of a probe having the sequence shown in FIG. 2(SEQ ID NO:3), or a complementary sequence, to target nucleic acid.

[0038] Hybridisation is generally followed by identification ofsuccessful hybridisation and isolation of nucleic acid which hashybridised to the probe, which may involve one or more steps of PCR. Itwill not usually be necessary to use a probe with the complete sequenceshown in any of these figures. Shorter fragments, particularly fragmentswith a sequence encoding the conserved TPR motifs may be used. Suitablesequences may include sequences encoding one or more of the TPR motifsshown in FIG. 4 (SEQ ID NOs:4-9).

[0039] Nucleic acids encoding or associated with the STRAP gene may alsobe used in methods of detecting the presence or absence of said gene ina human or non-human mammalian subject, said method comprising;

[0040] (a) bringing a sample of nucleic acid from said subject intocontact, under hybridizing conditions, with a polynucleotide of theinvention; and

[0041] (b) determining whether said polynucleotide has been able tohybridize to a homologous sequence in said nucleic acid.

[0042] The method may be performed using a polynucleotide primersuitable for use in a polymerase chain reaction (PCR), and thedetermining may be performed in conjunction with a second primer usingPCR such that a portion of the STRAP gene is amplified.

[0043] In one embodiment, the sample nucleic acid may be in the form ofwhole chromosomes, for example as a metaphase spread. The nucleic acidprobe or primer of the invention may be labelled with a fluorescentlabel to detect the chromosomal location of a STRAP gene in the spread.

[0044] In some instances, the determining step may include determiningthe sequence of the STRAP gene, when present, in the nucleic acidsample. As one alternative, restriction length fragment polymorphismsassociated with the gene may be established and the assay performed witha sample which has been digested with a restriction enzyme. Anothermethod of determining is via PCR length polymorphisms, for examplethrough variation in the sizes of introns. Other specific means ofdetermining hybridization are well known and routine in the art and mayalso be used.

[0045] As well as determining the presence of polymorphisms or mutationsin the STRAP sequence, the probes may also be used to determine whethermRNA encoding the STRAP gene is present in a cell or tissue.

[0046] Nucleic acid according to the present invention is obtainableusing one or more oligonucleotide probes or primers designed tohybridise with one or more fragments of the nucleic acid sequence shownin any of the figures, particularly fragments of relatively raresequence, based on codon usage or statistical analysis. A primerdesigned to hybridise with a fragment of the nucleic acid sequence shownin any of the figures may be used in conjunction with one or moreoligonucleotides designed to hybridise to a sequence in a cloning vectorwithin which target nucleic acid has been cloned, or in so-called “RACE”(rapid amplification of cDNA ends) in which cDNA's in a library areligated to an oligonucleotide linker and PCR is performed using a primerwhich hybridises with a sequence shown and a primer which hybridises tothe oligonucleotide linker.

[0047] Nucleic acid isolated and/or purified from one or more cells(e.g. human) or a nucleic acid library derived from nucleic acidisolated and/or purified from cells (e.g. a cDNA library derived frommRNA isolated from the cells), may be probed under conditions forselective hybridisation and/or subjected to a specific nucleic acidamplification reaction such as the polymerase chain reaction (PCR)(reviewed for instance in “PCR protocols; A Guide to Methods andApplications”, Eds. Innis et al, 1990, Academic Press, New York, Mulliset al, Cold Spring Harbor Symp. Quant. Biol., 51:263, (1987), Ehrlich(ed), PCR technology, Stockton Press, NY, 1989, and Ehrlich et al,Science, 252:1643-1650, (1991)). PCR comprises steps of denaturation oftemplate nucleic acid (if double-stranded), annealing of primer totarget, and polymerization. The nucleic acid probed or used as templatein the amplification reaction may be genomic DNA, cDNA or RNA. Otherspecific nucleic acid amplification techniques include stranddisplacement activation, the Qβ replicase system, the repair chainreaction, the ligase chain reaction and ligation activatedtranscription. For convenience, and because it is generally preferred,the term PCR is used herein in contexts where other nucleic acidamplification techniques may be applied by those skilled in the art.Unless the context requires otherwise, reference to PCR should be takento cover use of any suitable nucleic amplification reaction available inthe art.

[0048] In the context of cloning, it may be necessary for one or moregene fragments to be ligated to generate a full-length coding sequence.Also, where a full-length encoding nucleic acid molecule has not beenobtained, a smaller molecule representing part of the full molecule, maybe used to obtain full-length clones. Inserts may be prepared frompartial cDNA clones and used to screen cDNA libraries. The full-lengthclones isolated may be subcloned into expression vectors and activityassayed by transfection into suitable host cells, e.g. with a reporterplasmid.

[0049] A method may include hybridisation of one or more (e.g. two)probes or primers to target nucleic acid. Where the nucleic acid isdouble-stranded DNA, hybridisation will generally be preceded bydenaturation to produce single-stranded DNA. The hybridisation may be aspart of a PCR procedure, or as part of a probing procedure not involvingPCR. An example procedure would be a combination of PCR and lowstringency hybridisation. A screening procedure, chosen from the manyavailable to those skilled in the art, is used to identify successfulhybridisation events and isolated hybridised nucleic acid.

[0050] Binding of a probe to target nucleic acid (e.g. DNA) may bemeasured using any of a variety of techniques at the disposal of thoseskilled in the art. For instance, probes may be radioactively,fluorescently or enzymatically labelled. Other methods not employinglabelling of probe include examination of restriction fragment lengthpolymorphisms, amplification using PCR, RNase cleavage and allelespecific oligonucleotide probing. Probing may employ the standard.Southern blotting technique. For instance, DNA may be extracted fromcells and digested with different restriction enzymes. Restrictionfragments may then be separated by electrophoresis on an agarose gel,before denaturation and transfer to a nitrocellulose filter. Labelledprobe may be hybridised to the DNA fragments on the filter and bindingdetermined. DNA for probing may be prepared from RNA preparations fromcells.

[0051] Preliminary experiments may be performed by hybridising variousprobes under low stringency conditions to Southern blots of DNA digestedwith restriction enzymes. Suitable ‘conditions would be achieved’ when alarge number of hybridising fragments were obtained while the backgroundhybridisation was low. Using these conditions nucleic acid libraries,e.g. cDNA libraries representative of expressed sequences, may besearched. Those skilled in the art are well able to employ suitableconditions of the desired stringency for selective hybridisation, takinginto account factors such as oligonucleotide length and basecomposition, temperature and so on. On the basis of amino acid sequenceinformation, oligonucleotide probes or primers may be designed, takinginto account the degeneracy of the genetic code, and, where appropriate,codon usage of the organism from which the candidate nucleic acid isderived.

[0052] An oligonucleotide for use in nucleic acid amplification may haveabout 10 or fewer codons (e.g. 6, 7 or 8), i.e. be about 30 or fewernucleotides in length (e.g. 18, 21 or 24). Generally specific primersare upwards of 14 nucleotides in length, but need not be than 18-20.Those skilled in the art are well versed in the design of primers foruse processes such as PCR. Various techniques for synthesizingoligonucleotide primers are well known in the art, includingphosphotriester and phosphodiester synthesis methods.

[0053] Preferred amino acid sequences suitable for use in the design ofprobes or PCR primers may include sequences conserved (completely,substantially or partly) encoding the TPR motifs.

[0054] A further aspect of the present invention provides anoligonucleotide or polynucleotide fragment of the nucleotide sequenceshown in any of the figures herein providing nucleic acid according tothe present invention, or a complementary sequence, in particular foruse in a method of obtaining and/or screening nucleic acid. A sequencemay differ from any of the sequences shown by addition, substitution,insertion or deletion of one or more nucleotides, but preferably withoutabolition of ability to hybridise selectively with nucleic acid inaccordance with the present invention, that is wherein the degree ofsimilarity of the oligonucleotide or polynucleotide with one of thesequences given is sufficiently high.

[0055] In some preferred embodiments, oligonucleotides according to thepresent invention that are fragments of any of the sequences shown, areat least about 10 nucleotides in length, more preferably at least about15 nucleotides in length, more preferably at least about 20 nucleotidesin length. Such fragments themselves individually represent aspects ofthe present invention. Fragments and other oligonucleotides may be usedas primers or probes as discussed.

[0056] Further embodiments of oligonucleotides according to the presentinvention are anti-sense oligonucleotide sequences based on the nucleicacid sequences described herein. Anti-sense oligonucleotides may bedesigned to hybridise to the complementary sequence of nucleic acid,pre-mRNA or mature mRNA, interfering with the production of polypeptideencoded by a given DNA sequence (e.g. either native polypeptide or amutant form thereof), so that its expression is reduce or preventedaltogether. Anti-sense techniques may be used to target a codingsequence, a control sequence of a gene, e.g. in the 5′ flankingsequence, whereby the antisense oligonucleotides can interfere withcontrol sequences. Anti-sense oligonucleotides may be DNA or RNA and maybe of around 14-23 nucleotides, particularly around 15-18 nucleotides,in length. The construction of antisense sequences and their use isdescribed in Peyman and Ulman, Chemical Reviews, 90:543-584, (1990), andCrooke, Ann. Rev. Pharmacol. Toxicol., 32:329-376, (1992).

[0057] Any of the sequences disclosed in the figures herein may be usedto construct a probe for use in identification and isolation of apromoter from a genomic library containing a genomic STRAP gene.Techniques and conditions for such probing are well known in the art andare discussed elsewhere herein. To find minimal elements or motifsresponsible for stress and/or developmental regulation, restrictionenzyme or nucleases may be used to digest a nucleic acid molecule,followed by an appropriate assay (for example using a reporter gene suchas luciferase) to determine the sequence required.

[0058] A further aspect of the present invention provides a nucleic acidmolecule as described herein operably linked to a promoter or otherregulatory sequence.

[0059] By “promoter” is meant a sequence of nucleotides from whichtranscription may be initiated of DNA operably linked downstream (i.e.in the 3′ direction on the sense strand of double-stranded DNA).

[0060] “Operably linked” means joined as part of the same nucleic acidmolecule, suitably positioned and oriented for transcription to beinitiated from the promoter. DNA operably linked to a promoter is “undertranscriptional initiation regulation” of the promoter.

[0061] A further aspect of the present invention provides a polypeptidewhich has the amino acid sequence shown in FIG. 1 (SEQ ID NO:1 or SEQ IDNO:2), which may be in isolated and/or purified form, free orsubstantially free of material with which it is naturally associated,such as other polypeptides. If produced by expression in a prokaryoticcell) the polypeptide may be lacking in native glycosylation, e.g.unglycosylated. Polypeptides may of course be formulated with diluentsor adjuvants and still for practical purposes be isolated—for examplethe polypeptides will normally be mixed with gelatin or other carriersif used to coat microtitre plates for use in immunoassays. Polypeptidesmay be phosphorylated and/or acetylated.

[0062] Such a polypeptide is termed a STRAP polypeptide. This term alsoincludes amino acid sequence variants, alleles, derivatives and mutantsas well as active portions and fragments thereof.

[0063] The invention further provides active-portions and fragmentswhich comprises an epitope of said polypeptide. Unless otherwisespecified below, such portions and fragments are also referred to as apolypeptide of the present invention.

[0064] Polypeptides which are amino acid sequence variants, alleles,derivatives or mutants are also provided by the present invention. Apolypeptide which is a variant, allele, derivative or mutant may have anamino acid sequence which differs from that given in a figure herein byone or more of addition, substitution, deletion and insertion of one ormore amino acids. Preferred such polypeptides have STRAP function, thatis to say have one or more of the following properties: immunologicalcross-reactivity with an antibody reactive the polypeptide for which thesequence is given in a figure herein; sharing an epitope with thepolypeptide for which the amino acid sequence is shown in a figureherein (as determined for example by immunological cross-reactivitybetween the two polypeptides); a biological activity which is inhibitedby an antibody raised against the polypeptide whose sequence is shown ina figure herein; ability to bind with p300 and/or JMY. Alteration ofsequence may change the nature and/or level of activity and/or stabilityof the STRAP polypeptide.

[0065] Polypeptides of the invention may be modified for example by theaddition of histidine residues to assist their purification or by theaddition of a signal sequence to promote their secretion from a cell.

[0066] A polypeptide which is an amino acid sequence variant, allele,derivative or mutant of the amino acid sequence shown in a figure hereinmay comprise an amino acid sequence which shares greater than about 35%sequence identity with the sequence shown, greater than about 40%,greater than about 50%, greater than about 60%, greater than about 70%,greater than about 80%, greater than about 90% or greater than about95%. The sequence may share greater than about 60% similarity, greaterthan about 70% similarity, greater than about 80% similarity or greaterthan about 90% similarity with the amino acid sequence shown in therelevant figure.

[0067] Amino acid similarity is generally defined with reference to thealgorithm GAP (Genetics Computer Group, Madison, Wis.) as noted above,or the TBLASTN program, of Altschul et al. (1990) J. Mol. Biol. 215:403-10. Similarity allows for “conservative variation”, i.e.substitution of one hydrophobic residue such as isoleucine, valine,leucine or methionine for another, or the substitution of one polarresidue for another, such as arginine for iysine, glutamic for asparticacid, or glutamine for asparagine. Particular amino acid sequencevariants may differ from that shown in a figure herein by insertion,addition, substitution or deletion of 1 amino acid, 2, 3, 4, 5-10, 10-2020-30, 30-50, 50-100, 100-150, or more than 150 amino acids.

[0068] Sequence comparison may be made over the full-length of therelevant sequence shown herein, or may more preferably be over acontiguous sequence of about or greater than about 20, 25, 30, 33, 40,50, 67, 133, 167, 200, 233, 267, 300, 333, 400, or more amino acids,compared with the relevant amino acid sequence as the case may be.

[0069] Preferred such polypeptides include those which are encoded bythe STRAP gene of other mammals, particularly primates and mostparticularly man, as well as fragments of such polypeptides, suchfragments being those as defined above.

[0070] The primary sequence of the STRAP protein will be substantiallysimilar to that of FIG. 1 (SEQ ID NO:1 or SEQ ID NO:2) and may bedetermined by routine techniques available to those of skill in the art.In essence, such techniques comprise using polynucleotides of thepresent invention as probes to recover and to determine the sequence ofthe STRAP gene in other species. Human STRAP is shown in FIG. 1B (SEQ IDNO:2), and was obtained in the light of the present invention byanalysis of publicly available sequence databases. The databases do notidentify that this protein as such, nor identify the function that isdisclosed herein.

[0071] A wide variety of techniques are available for this, for examplePCR amplification and cloning of the gene using a suitable source ofmRNA (e.g. from an embryo or an actively dividing differentiated ortumour cell), or by methods comprising obtaining a cDNA library from themammal, e.g a cDNA library from one of the above-mentioned sources,probing said library with a polynucleotide of the invention understringent conditions, and recovering a cDNA encoding all or part of theSTRAP protein of that mammal. Where a partial cDNA is obtained, the fulllength coding sequence may be determined by primer extension techniques.

[0072] The present invention also includes peptides which include orconsist of fragments of a polypeptide of the invention.

[0073] The present inventors have also identified regions in the JMY andp300 sequence which interact with STRAP. A peptide consisting of such aregion and nucleic acid encoding such a peptide are further aspects ofthe present invention.

[0074] Regions of JMY which interact with STRAP include residues 683 to983 and regions of p300 which interact with STRAP include residues 1 to595 and residues 1572 to 1921. The sequence of JMY is available asGenBank accession no. AAF 17555 and the sequence of p300 available asGenBank accession number XP010013.

[0075] “p300” refers to a family member of the p300/CBP family ofco-activators which have histone acetyltransferase activity p300 isdescribed for example by Eckner et al, 1994 and CBP by Bannister andKouzarides, 1996. For the purposes of the present invention, referenceto “p300” or “p300 polypeptide” refers to human allelic and syntheticvariants of p300 or CBP, and to other mammalian variants and allelic andsynthetic variants thereof, as well as fragments of said human andmammalian forms of p300 or CBP. Synthetic variants include those whichhave at least 80%, preferably at least 90%, homology to p300. Morepreferably such variants correspond to the sequence of p300 but have oneor more, e.g. from 1 to 10, such as from 1 to 5, substitutions,deletions or insertions of amino acids. Fragments of p300 and itsvariants are preferably at least 20, more preferably at least 50 andmost preferably at least 200 amino acids in size. The p300 molecule willhowever retain the ability to physically associate in vivo with STRAP.

[0076] Preferably, the p300 used in assays of the present invention willalso retain the ability to interact with the tumour suppressor moleculep53, as described in the accompanying examples and by Lill et al, 1997.

[0077] For the purposes of the present invention, the precise form andstructure of a p300 protein or fragment thereof may be varied by thoseof skill in the art, having regard to the particular assay format to beused.

[0078] “JMY” refers to any family member of the JMY family ofco-activators which bind the p300/CBP co-activator complex and aredisclosed in PCT/GB98/03152. For the purposes of the present invention,reference to “JMY” or “JMY polypeptide” refers to human allelic andsynthetic variants of JMY, and to other mammalian variants and allelicand synthetic variants thereof, as well as fragments of said human andmammalian forms of JMY. Synthetic variants include those which have atleast 80%, preferably at least 90%, homology to JMY. More preferablysuch variants correspond to the sequence of JMY but have one or more,e.g. from 1 to 10, such as from 1 to 5, substitutions, deletions orinsertions of amino acids. Fragments of JMY and its variants arepreferably at least 20, more preferably at least 50 and most preferablyat least 200 amino acids in size. The JMY molecule will however retainthe ability to physically associate in vivo with STRAP and/or p300.

[0079] For the purposes of the present invention, the precise form andstructure of a JMY protein or fragment thereof may be varied by those ofskill in the art, having regard to the particular assay format to beused.

[0080] “p53” refers to the tumour suppressor gene or its encoded aminoacid sequence of as reported, for example, by Matlashewski et al (EMBOJ. 13; 3257-62, 1984) or Lamb and Crawford (Mol. Cell. Biol. 5; 1379-85,1986). These sequences are available on Genbank. Wild-type human p53protein includes a proline/arginine polymorphism at amino acid 72,reflecting a corresponding polymorphism in the gene.

[0081] The skilled person can use the techniques described herein andothers well known in the art to produce large amounts of peptides, forinstance by expression from encoding nucleic acid.

[0082] Peptides can also be generated wholly or partly by chemicalsynthesis. The compounds of the present invention can be readilyprepared according to well-established, standard liquid or, preferably,solid-phase peptide synthesis methods, general descriptions of which arebroadly available (see, for example, in J. M. Stewart and J. D. Young,Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Company,Rockford, Ill. (1984), in M. Bodanzsky and A. Bodanzsky, The Practice ofPeptide Synthesis, Springer Verlag, New York (1984); and AppliedBiosystems 430A Users Manual, ABI Inc., Foster City, Calif.).

[0083] The present invention also includes active portions, fragments,derivatives and functional mimetics of the polypeptides of theinvention. An “active portion” of a polypeptide means a peptide which isless than said full length polypeptide, but which retains a biologicalactivity, such as binding to p300 and/or JMY. Thus an active portion ofthe STRAP polypeptide may include amino acids 1 to 123 or amino acids123 to 205 which bind JMY and/or may include amino acids 206 to 440,which bind p300. Such an active fragment may be included as part of afusion protein, e.g. including a binding portion for a different ligandwhich may confer on the molecule a different binding specificity.

[0084] Active portions may also include those which are phosphorylatedand/or acetylated, particularly in a cell-cycle specific manner.

[0085] A “fragment” of a polypeptide generally means a stretch of aminoacid residues of at least about five contiguous amino acids, often atleast about seven contiguous amino acids, typically at least about ninecontiguous amino acids, more preferably at least about 13 contiguousamino acids, and, more preferably, at least about 20 to 30 or morecontiguous amino acids. Fragments of the STRAP polypeptide sequence mayinclude antigenic determinants or epitopes useful for raising antibodiesto a portion of the amino acid sequence. Alanine scans are commonly usedto find and refine peptide motifs within polypeptides, this involvingthe systematic replacement of each residue in turn with the amino acidalanine, followed by an assessment of biological activity.

[0086] Preferred fragments of STRAP include those which contain any ofthe following amino acid sequences shown in FIG. 1 (SEQ ID NO: 1 or SEQID NO:2): residues 1 to 123, residues 122 to 205, residues 206 to 440,which may be used for instance in raising or isolating antibodies.Variant and derivative peptides, peptides which have an amino acidsequence which differs from one of these sequences by way of addition,insertion, deletion or substitution of one or more amino acids are alsoprovided by the present invention, generally with the proviso that thevariant or derivative peptide is bound by an antibody or other specificbinding member which binds one of the peptides whose sequence is shown.A peptide which is a variant or derivative of one of the shown peptidesmay compete with the shown peptide for binding to a specific bindingmember, such as an antibody or antigen-binding fragment thereof.

[0087] Where additional amino acids are included in a peptide, these maybe heterologous or foreign to the polypeptide of the invention, and thepeptide may be about 20, 25, 30 or 35 amino acids in length. A peptideaccording to this aspect may be included within a larger fusion protein,particularly where the peptide is fused to a non-STRAP (i.e.heterologous or foreign) sequence, such as a polypeptide or proteindomain.

[0088] A “derivative” of a polypeptide or a fragment thereof may includea polypeptide modified by varying the amino acid sequence of theprotein, e.g. by manipulation of the nucleic acid encoding the proteinor by altering the protein itself. Such derivatives of the natural aminoacid sequence may involve one or more of insertion, addition, deletionor substitution of one or more amino acids, which may be withoutfundamentally altering the qualitative nature of biological activity ofthe wild type polypeptide.

[0089] Also encompassed within the scope of the present invention arefunctional mimetics of active fragments of the STRAP polypeptidesprovided (including alleles, mutants, derivatives and variants). Theterm “functional mimetic” means a substance which may not contain anactive portion of the relevant amino acid sequence, and probably is nota peptide at all, but which retains in qualitative terms a biologicalactivity of natural STRAP polypeptide. The design and screening ofcandidate mimetics is described in detail below.

[0090] A polypeptide according to the present invention may be isolatedand/or purified (e.g. using an antibody) for instance after productionby expression from encoding nucleic acid (for which see below). Thus, apolypeptide may be provided free or substantially free from contaminantswith which it is naturally associated (if it is a naturally-occurringpolypeptide). A polypeptide may be provided free or substantially freeof other polypeptides.

[0091] Polypeptides according to the present invention may be generatedwholly or partly by chemical synthesis. The isolated and/or purifiedpolypeptide may be used in formulation of a composition, which mayinclude at least one additional component, for example a pharmaceuticalcomposition including a pharmaceutically acceptable excipient, vehicleor carrier. A composition including a polypeptide according to theinvention may be used in prophylactic and/or therapeutic treatment asdiscussed below.

[0092] A polypeptide, peptide, allele, mutant, derivative or variantaccording to the present invention may be used as an immunogen orotherwise in obtaining specific antibodies. Antibodies are useful inpurification and other manipulation of polypeptides and peptides andtherapeutic contexts. This is discussed further below.

[0093] A polypeptide of the invention may be labelled with a revealinglabel. The revealing label may be any suitable label which allows thepolypeptide to be detected. Suitable labels include radioisotopes, e.g.125I, enzymes, antibodies, polynucleotides and linkers such as biotin.Labelled polypeptides of the invention may be used in diagnosticprocedures such as immunoassays in order to determine the amount of apolypeptide of the invention in a sample. Polypeptides or labelledpolypeptides of the invention may also be used in serological or cellmediated immune assays for the detection of immune reactivity to saidpolypeptides in animals and humans using standard protocols.

[0094] A polypeptide or labelled polypeptide of the invention orfragment thereof may also be fixed to a solid phase, for example thesurface of an immunoassay well or dipstick.

[0095] Such labelled and/or immobilized polypeptides may be packagedinto kits in a suitable container along with suitable reagents,controls, instructions and the like.

[0096] Such polypeptides and kits may be used in methods of detection ofantibodies to such polypeptides present in a sample or active portionsor fragments thereof by immunoassay.

[0097] Immunoassay methods are well known in the art and will generallycomprise:

[0098] (a) providing a polypeptide comprising an epitope bindable by anantibody against said protein;

[0099] (b) incubating a biological sample with said polypeptide underconditions which allow for the formation of an antibody-antigen complex;and

[0100] (c) determining whether antibody-antigen complex comprising saidpolypeptide is formed.

[0101] A polypeptide according to the present invention may be used inscreening for molecules which affect or modulate its activity orfunction, e.g its interactions with p300 co-activator complex and/or itseffect on p53 activity. Such molecules may interact with the N terminalregion (residues 1 to 123), a region between amino acids 124 to 205 or aC terminal (residues 206 to 440) region of STRAP or with one or moreregions of JMY and/or p300 which bind to STRAP, and may be useful in atherapeutic (including prophylactic) context.

[0102] It is well known that pharmaceutical research leading to theidentification of a new drug may involve the screening of very largenumbers of candidate substances, both before and even after a leadcompound has been found. This is one factor which makes pharmaceuticalresearch very expensive and time-consuming. Means for assisting in thescreening process can have considerable commercial importance andutility. Such means for screening for substances potentially useful inmodulating (i.e. activating or reducing) the activity of p53 andtreating or preventing p53 induced apoptosis are provided bypolypeptides according to the present invention.

[0103] Substances identified as modulators of the interactions describedherein are extremely useful in the modulation of a range of stressrelated p53 activities since they provide basis for design andinvestigation of therapeutics for in vivo use. Furthermore, they may beuseful in any of a number of conditions in which p53 activity isundesirable, including cancer-therapy genotoxicity, p53 dependentneuronal death in the central nervous system (i.e. brain or spinal cordinjury), preservation of tissues or organs prior to transplant,preparation of host for bone marrow transplant, reducing neuronal damageduring seizures, and suppression of cell aging. As noted elsewhere,STRAP and fragments thereof may also be useful in combating any of theseconditions and disorders.

[0104] In various further aspects the present invention relates toscreening and assay methods and means, and substances identifiedthereby.

[0105] The identification of the polypeptide expressed by the STRAP geneenables assays to be developed to identify further cellular proteinswith which the polypeptide is associated, in addition to p300 and JMY.For example, polypeptides of the present invention may be required in aregulatory pathway in which their function is to interact with otherfactors which in turn promote or maintain essential cellular functionsassociated with cell cycle control. The polypeptides of the presentinvention may be used in two-hybrid assays as described below todetermine cellular factors with which they become associated.

[0106] Assay methods may therefore be for substances or agents whichinteract with or bind a polypeptide of the invention and/or modulate oneor more of its activities.

[0107] Further aspects of the present invention provide the use of aSTRAP polypeptide or peptide (particularly a fragment of a polypeptideof the invention as disclosed), and/or encoding nucleic acid therefor,in screening or searching for and/or obtaining/identifying a substance,e.g. peptide or chemical compound, which interacts and/or binds with theSTRAP polypeptide or peptide and/or interferes with the interactionbetween STRAP and JMY and/or STRAP and p300 and which is therefore acandidate modulator of the function or activity of the p300 co-factorcomplex. Such a substance may be useful in modulating the activity ofp53, for example by means of modulating the half life of p53 and/ormodulating the activity of target genes which execute the p53 stressresponse.

[0108] For instance, a method according to one aspect of the inventionincludes providing a polypeptide or peptide of the invention andbringing it into contact with a substance, which contact may result inbinding between the polypeptide or peptide and the substance. Bindingmay be determined by any of a number of techniques available in the art,both qualitative and quantitative.

[0109] A method of screening for a substance which modulates the bindingactivity of a STRAP polypeptide may include contacting one or more testsubstances with the STRAP polypeptide in a suitable reaction medium,testing the binding activity of the treated polypeptide and comparingthat activity with the binding activity of the STRAP polypeptide incomparable reaction medium untreated with the test substance orsubstances. A difference in binding activity between the treated anduntreated polypeptides is indicative of a modulating effect of therelevant test substance or substances.

[0110] Corresponding aspects of the present invention relate to methodsof screening for a substance which modulates the binding activity of apolypeptide consisting of residues 683 to 983 of JMY or a polypeptideconsisting of residues 1 to 595 or 1572 to 1921 of p300.

[0111] In various aspects the present invention is concerned withprovision of assays for substances which inhibit interaction between apolypeptide of the invention and one or more of JMY and p300.

[0112] One aspect of the present invention provides an assay methodwhich includes:

[0113] (i) bringing into contact a STRAP polypeptide according to theinvention and a putative binding molecule or other test substance; and

[0114] (ii) determining interaction or binding between the STRAPpolypeptide and the test substance.

[0115] A substance which interacts with a STRAP polypeptide or peptideof the invention may be isolated and/or purified, manufactured and/orused to modulate its activity as discussed.

[0116] A further aspect of the present invention provides an assaymethod for screening for a substance which modulates the binding of JMYand STRAP, including:

[0117] (i) bringing a STRAP polypeptide into contact with a JMYpolypeptide in the presence of one or more test substances; and

[0118] (ii) determining the binding of the STRAP polypeptide to the JMYpolypeptide.

[0119] The STRAP polypeptide may be brought into contact with the JMYpolypeptide in the presence of a p300 polypeptide.

[0120] Another aspect of the present invention provides an assay methodfor screening for a substance which modulates the binding of p300 andSTRAP, including:

[0121] (i) bringing a STRAP polypeptide into contact with a p300polypeptide in the presence of one or more test substances; and

[0122] (ii) determining the binding of the STRAP polypeptide to the p300polypeptide.

[0123] The STRAP polypeptide may be brought into contact with the p300polypeptide in the presence of a JMY polypeptide.

[0124] Another aspect of the present invention provides an assay methodfor screening for a substance which modulates the binding of JMY, p300and STRAP, including:

[0125] (i) bringing a STRAP polypeptide into contact with a p300polypeptide and a JMY polypeptide in the presence of one or more testsubstances; and

[0126] (ii) determining the binding of the STRAP polypeptide to the p300polypeptide and the JMY polypeptide.

[0127] An assay may be carried out under conditions in which in theabsence of the test substance being an inhibitor, the STRAP polypeptidebinds to the JMY or p300 polypeptide.

[0128] In assays of the present invention, the binding of a JMY and/orp300 polypeptide to a STRAP polypeptide may be determined in thepresence and absence of the test substance. A difference in bindingactivity in the presence of test substance is indicative of a modulatingeffect of the relevant test substance or substances.

[0129] An assay method may comprise determining the p53 stress responsein the presence and/or absence of said test substance as describedherein.

[0130] As mentioned above, it is not necessary to use the entireproteins for assays of the invention which test for binding between twomolecules. Fragments may be generated and used in any suitable way knownto those of skill in the art. Suitable ways of generating fragmentsinclude, but are not limited to, recombinant expression of a fragmentfrom encoding DNA. Such fragments may be generated by taking encodingDNA, identifying suitable restriction enzyme recognition sites eitherside of the portion to be expressed, and cutting out said portion fromthe DNA. The portion may then be operably linked to a suitable promoterin a standard commercially available expression system. Anotherrecombinant approach is to amplify the relevant portion of the DNA withsuitable PCR primers. Small fragments (e.g. up to about 20 or 30 aminoacids) may also be generated using peptide synthesis methods which arewell known in the art.

[0131] The precise format of the assay of the invention may be varied bythose of skill in the art using routine skill and knowledge. Forexample, the interaction between the polypeptides may be studied invitro by labelling one with a detectable label and bringing it intocontact with the other which has been immobilised on a solid support.Suitable detectable labels include 35S-methionine which may beincorporated into recombinantly produced peptides and polypeptides.Recombinantly produced peptides and polypeptides may also be expressedas a fusion protein containing an epitope which can be labelled with anantibody.

[0132] Fusion proteins may be generated that incorporate six histidineresidues at either the N-terminus or C-terminus of the recombinantprotein. Such a histidine tag may be used for purification of theprotein by using commercially available columns which contain a metalion, either nickel or cobalt (Clontech, Palo Alto, Calif., USA). Thesetags also serve for detecting the protein using commercially availablemonoclonal antibodies directed against the six histidine residues(Clontech, Palo Alto, Calif., USA).

[0133] The protein which is immobilized on a solid support may beimmobilized using an antibody against that protein bound to a solidsupport or via other technologies which are known per se. A preferred invitro interaction may utilise a fusion protein includingglutathione-S-transferase (GST). This may be immobilized on glutathioneagarose beads. In an in vitro assay format of the type described above atest compound can be assayed by determining its ability to diminish theamount of labelled peptide or polypeptide which binds to the immobilizedGST-fusion polypeptide. This may be determined by fractionating theglutathione-agarose beads by SDS-polyacrylamide gel electrophoresis.Alternatively, the beads may be rinsed to remove unbound protein and theamount of protein which has bound can be determined by counting theamount of label present in, for example, a suitable scintillationcounter.

[0134] In an alternative mode, one of STRAP polypeptide and p300 or JMYpolypeptide may be labelled with a fluorescent donor moiety and theother labelled with an acceptor which is capable of reducing theemission from the donor. This allows an assay according to the inventionto be conducted by fluorescence resonance energy transfer (FRET). Inthis mode, the fluorescence signal of the donor will be altered whenSTRAP and p300 or JMY interact. The presence of a candidate modulatorcompound which modulates the interaction will increase the amount ofunaltered fluorescence signal of the donor.

[0135] FRET is a technique known per se in the art and thus the precisedonor and acceptor molecules and the means by which they are linked toSTRAP and p300 or JMY may be accomplished by reference to theliterature.

[0136] Suitable fluorescent donor moieties are those capable oftransferring fluorogenic energy to another fluorogenic molecule or partof a compound and include, but are not limited to, coumarins and relateddyes such as fluoresceins, rhodols and rhodamines, resorufins, cyaninedyes, bimanes, acridines, isoindoles, dansyl dyes, aminophthalichydrazines such as luminol and isoluminol derivatives,aminophthalimides, aminonaphthalimides, aminobenzofurans,aminoquinolines, dicyanohydroquinones, and europium and terbiumcomplexes and related compounds.

[0137] Suitable acceptors include, but are not limited to, coumarins andrelated fluorophores, xanthenes such as fluoresceins, rhodols andrhodamines, resorufins, cyanines, difluoroboradiazaindacenes, andphthalocyanines.

[0138] A preferred donor is fluorescein and preferred acceptors includerhodamine and carbocyanine. The isothiocyanate derivatives of thesefluorescein and rhodamine, available from Aldrich Chemical Company Ltd,Gillingham, Dorset, UK, may be used to label STRAP or JMY or p300/CBP.For attachment of carbocyanine, see for example Guo et al, J. Biol.Chem., 270; 27562-8, 1995.

[0139] Another assay format is dissociation enhanced lanthanidefluorescent immunoassay (DELFIA) (Ogata et al, 1992). This is a solidphase based system for measuring the interaction of two macromolecules.Typically one molecule (either STRAP or JMY or p300) is immobilised tothe surface of a multi well plate and the other molecule is added insolution to this. Detection of the bound partner is achieved by using alabel consisting of a chelate of a rare earth metal. This label can bedirectly attached to the interacting molecule or may be introduced tothe complex via an antibody to the molecule or to the molecules epitopetag. Alternatively, the molecule may be attached to biotin and astreptavidin-rare earth chelate used as the label. The rare earth usedin the label may be europium, samarium, terbium or dysprosium. Afterwashing to remove unbound label, a detergent containing low pH buffer isadded to dissociate the rare earth metal from the chelate. The highlyfluorescent metal ions are then quantitated by time resolvedfluorimetry. A number of labelled reagents are commercially availablefor this technique, including streptavidin, antibodies againstglutathione-S-transferase and against hexahistidine.

[0140] An assay according to the present invention may also take theform of an in vivo assay. The in vivo assay may be performed in a cellline such as a yeast strain in which the relevant polypeptides orpeptides are expressed from one or more vectors introduced into thecell.

[0141] In vivo assays may also take the form of two-hybrid assayswherein STRAP and p300 or JMY are expressed as fusion proteins, onebeing a fusion protein comprising a DNA binding domain (DBD), such asthe yeast GAL4 binding domain, and the other being a fusion proteincomprising an activation domain, such as that from GAL4 or VP16. In sucha case the host cell (which again may be bacterial, yeast, insect ormammalian, particularly yeast or mammalian) will carry a reporter geneconstruct with a promoter comprising a DNA binding elements compatiblewith the DBD.

[0142] STRAP and JMY or p300 and the reporter gene, may be introducedinto the cell and expressed transiently or stably.

[0143] Alternatively, assays of the invention may be conducted byutilizing the ability of a STRAP-p300 complex (including JMY) to mediatethe activation of a reporter gene or to induce a cellular response in acell, particularly apoptosis. For example, a number of transcriptionfactors, including the glucocorticoid receptor (GR) and E2F-1, are knownto be regulated by p300/CBP, as is p53. We have found that theregulation of such factors is enhanced by STRAP. Further, we have foundthat p53-mediated apoptosis is enhanced by the presence of STRAP.

[0144] Thus assays of the invention include an assay for a modulator ofSTRAP-p300 complex formation which comprises:

[0145] a) providing STRAP, p300 and JMY together with a regulatoryfactor which is a target for p300, in the presence of a putativemodulator and a reporter gene which comprises a target promoter for saidregulatory factor; and

[0146] b) measuring the modulation of transcription of the reporter genecaused by the presence of said modulator.

[0147] The regulatory factor includes GR for which suitable promotersinclude promoters which contain a GRE such as c-myc and the MMLV LTR;E2F-1 for which suitable promoters include cyclin A, cyclin E, tyrosineamino transferase and the E2F-1 gene promoter; p53 for which suitablepromoters include Bax, Waf1/Cip, Gadd45 and cyclin G; oestrogen receptor(ER) for which suitable promoters include progesterone receptor andPS-2; and other nuclear receptors and promoters containing recognitionelements of this type. Suitable reporter genes operably linked to thepromoter include chloramphenicol acetyl transferase, luciferase, greenfluorescent protein and β-galactosidase. In the case of ER, a the 13base palindromic estrogen response element (ERE) may be included in thepromoter of a reporter construct to provide a suitable reporter gene. Inan alternative embodiment, the assay may be conducted in a cell lackingwild-type p53 and which undergoes apoptosis in the presence of p53. Suchcells include SAOS-2 cells.

[0148] In this format the assay will be conducted by supplying to thecell expression vector(s) encoding STRAP, JMY, p300/CBP and wild typep53, treating said cells with a putative modulator and measuring theeffect of the modulator on apoptosis of the cells. Apoptosis may also bemeasured in an analogous manner in cell lines with wild type p53 whereinapoptosis is enhanced by the presence of, for example, excess STRAP.

[0149] Assays will be run with suitable controls routine to those ofskill in the art.

[0150] Accordingly, another aspect of the present invention is asubstance obtainable using an assay method as described herein.

[0151] Such a substance may include polypeptide, antibody, peptide,nucleic acid molecule, small molecule or other pharmaceutically usefulcompound. In some embodiments of this aspect of the invention, apolypeptide has less than 900 residues and/or does not include the fulllength JMY and p300 sequences.

[0152] Combinatorial library technology (Schultz, J S (1996) Biotechnol.Prog. 12:729-743) provides an efficient way of testing a potentiallyvast number of different substances for ability to modulate activity ofa polypeptide. Prior to, or as well as, being screened for modulation ofactivity, test substances may be screened for ability to interact withthe polypeptide, e.g. in a yeast two-hybrid system (which requires thatboth the polypeptide and the test substance can be expressed in yeastfrom encoding nucleic acid). This may be used as a coarse screen priorto testing a substance for actual ability to modulate activity of thepolypeptide.

[0153] The amount of test substance or compound which may be added to anassay of the invention will normally be determined by trial and errordepending upon the type of compound used. Typically, from about 0.01 to100 μM concentrations of putative inhibitor compound may be used, forexample from 0.1 to 10 μM. Greater concentrations may be used when apeptide is the test substance.

[0154] Compounds which may be used may be natural or synthetic chemicalcompounds used in drug screening programmes. Extracts of plants whichcontain several characterised or uncharacterised components may also beused. A further class of putative inhibitor compounds can be derivedfrom the STRAP polypeptide or the JMY polypeptide and/or p300polypeptide which to which it binds. Peptide fragments of from 5 to 40amino acids, for example from 6 to 10 amino acids from the region of therelevant polypeptide responsible for interaction, may be tested fortheir ability to disrupt such interaction.

[0155] Other candidate inhibitor compounds may be based on modelling the3-dimensional structure of a polypeptide or peptide fragment and usingrational drug design to provide potential inhibitor compounds withparticular molecular shape, size and charge characteristics.

[0156] Following identification of a substance which modulates oraffects polypeptide activity, the substance may be investigated further.Furthermore, it may be manufactured and/or used in preparation, i.e.manufacture or formulation, of a composition such as a medicament,pharmaceutical composition or drug. These may be administered toindividuals.

[0157] Thus, the present invention extends in various aspects not onlyto a substance identified as a modulator of polypeptide activity, inaccordance with what is disclosed herein, but also a pharmaceuticalcomposition, medicament, drug or other composition comprising such asubstance, a method comprising administration of such a composition to apatient, e.g. for treatment (which may include preventative treatment)of a condition related to stress induced p53 dependent cell apoptosis,such as DNA damage (for example caused by UV radiation), cancer-therapygenotoxicity (for example, caused by chemo- or radiation therapy), p53dependent neuronal death in the central nervous system (i.e. brain orspinal cord injury), preservation of tissues or organs prior totransplant, preparation of host for bone marrow transplant, reducingneuronal damage during seizures and suppression of cell aging, use ofsuch a substance in manufacture of a composition for administration,e.g. for treatment of a condition related to p53 dependent cellapoptosis (such as hyperthermia, hypoxia, stroke, ischemia, acuteinflammation, burn or cell aging), and a method of making apharmaceutical composition comprising admixing such a substance with apharmaceutically acceptable excipient, vehicle or carrier, andoptionally other ingredients.

[0158] Cancer therapy includes radio- and chemotherapy.

[0159] A substance identified using as a modulator of polypeptide orpromoter function may be peptide or non-peptide in nature. Non-peptide“small molecules” are often preferred for many in vivo pharmaceuticaluses. Accordingly, a mimetic or mimic of the substance (particularly ifa peptide) may be designed for pharmaceutical use. The designing ofmimetics to a known pharmaceutically active compound is a known approachto the development of pharmaceuticals based on a “lead” compound. Thismight be desirable where the active compound is difficult or expensiveto synthesise or where it is unsuitable for a particular method ofadministration, e.g. peptides are not well suited as active agents fororal compositions as they tend to be quickly degraded by proteases inthe alimentary canal. Mimetic design, synthesis and testing may be usedto avoid randomly screening large number of molecules for a targetproperty.

[0160] There are several steps commonly taken in the design of a mimeticfrom a compound having a given target property. Firstly, the particularparts of the compound that are critical and/or important in determiningthe target property are determined. In the case of a peptide, this canbe done by systematically varying the amino acid residues in thepeptide, e.g. by substituting each residue in turn. These parts orresidues constituting the active region of the compound are known as its“pharmacophore”.

[0161] Once the pharmacophore has been found, its structure is modelledto according its physical properties, e.g. stereochemistry, bonding,size and/or charge, using data from a range of sources, e.g.spectroscopic techniques, X-ray diffraction data and NMR. Computationalanalysis, similarity mapping (which models the charge and/or volume of apharmacophore, rather than the bonding between atoms) and othertechniques can be used in this modelling process.

[0162] In a variant of this approach, the three-dimensional structure ofthe ligand and its binding partner are modelled. This can be especiallyuseful where the ligand and/or binding partner change conformation onbinding, allowing the model to take account of this the design of themimetic.

[0163] A template molecule is then selected onto which chemical groupswhich mimic the pharmacophore can be grafted. The template molecule andthe chemical groups grafted on to it can conveniently be selected sothat the mimetic is easy to synthesise, is likely to bepharmacologically acceptable, and does not degrade in vivo, whileretaining the biological activity of the lead compound. The mimetic ormimetics found by this approach can then be screened to see whether theyhave the target property, or to what extent they exhibit it. Furtheroptimisation or modification can then be carried out to arrive at one ormore final mimetics for in vivo or clinical testing.

[0164] Mimetics of substances identified as having ability to modulateSTRAP polypeptide activity using a screening method as disclosed hereinare included within the scope of the present invention. A polypeptide,peptide or substance able to modulate activity of a STRAP polypeptideaccording to the present invention may be provided in a kit, e.g. sealedin a suitable container which protects its contents from the externalenvironment. Such a kit may include instructions for use.

[0165] A convenient way of producing a polypeptide according to thepresent invention is to express nucleic acid encoding it, by use of thenucleic acid in an expression system. Accordingly, the present inventionalso encompasses a method of making a polypeptide (as disclosed), themethod including expression from nucleic acid encoding the polypeptide(generally nucleic acid according to the invention). This mayconveniently be achieved by growing a host cell in culture, containingsuch a vector, under appropriate conditions which cause or allowexpression of the polypeptide. Polypeptides may also be expressed in invitro systems, such as reticulocyte lysate.

[0166] Systems for cloning and expression of a polypeptide in a varietyof different host cells are well known. Suitable host cells includebacteria, eukaryotic cells such as mammalian and yeast, and baculovirussystems. Mammalian cell lines available in the art for expression of aheterologous polypeptide include Chinese hamster ovary cells, HeLacells, baby hamster kidney cells, COS cells and many others. A common,preferred bacterial host is E. coli. Suitable vectors can be chosen orconstructed, containing appropriate regulatory sequences, includingpromoter sequences, terminator fragments, polyadenylation sequences,enhancer sequences, marker genes and other sequences as appropriate.Vectors may be plasmids, viral e.g. phage, or phagemid, as appropriate.For further details see, for example, Molecular Cloning: a LaboratoryManual: 2nd edition, Sambrook et al., 1989, Cold Spring HarborLaboratory Press. Many known techniques and protocols for manipulationof nucleic acid, for example in preparation of nucleic acid constructs,mutagenesis, sequencing, introduction of DNA into cells and geneexpression, and analysis of proteins, are described in detail in CurrentProtocols in Molecular Biology, Ausubel et al. eds., John Wiley & Sons,1992.

[0167] Thus, a further aspect of the present invention provides a hostcell containing nucleic acid as disclosed herein. The nucleic acid ofthe invention may be integrated into the genome (e.g. chromosome) of thehost cell. Integration may be promoted by inclusion of sequences whichpromote recombination with the genome, in accordance with standardtechniques. The nucleic acid may be on an extra-chromosomal vectorwithin the cell.

[0168] A still further aspect provides a method which includesintroducing the nucleic acid into a host cell. The introduction, whichmay (particularly for in vitro introduction) be generally referred towithout limitation as “transformation”, may employ any availabletechnique. For eukaryotic cells, suitable techniques may include calciumphosphate transfection, DEAE-Dextran, electroporation, liposome-mediatedtransfection and transduction using retrovirus or other virus, e.g.vaccinia or, for insect cells, baculovirus. For bacterial cells,suitable techniques may include calcium chloride transformation,electroporation and transfection using bacteriophage.

[0169] Marker genes such as antibiotic resistance or sensitivity genesmay be used in identifying clones containing nucleic acid of interest,as is well known in the art.

[0170] The introduction may be followed by causing or allowingexpression from the nucleic acid, e.g. by culturing host cells (whichmay include cells actually transformed although more likely the cellswill be descendants of the transformed cells) under conditions forexpression of the gene, so that the encoded polypeptide is produced. Ifthe polypeptide is expressed coupled to an appropriate signal leaderpeptide it may be secreted from the cell into the culture medium.Following production by expression, a polypeptide may be isolated and/orpurified from the host cell and/or culture medium, as the case may be,and subsequently used as desired, e.g. in the formulation of acomposition which may include one or more additional components, such asa pharmaceutical composition which includes one or more pharmaceuticallyacceptable excipients, vehicles or carriers (e.g. see below).

[0171] Introduction of nucleic acid may take place in vivo by way ofgene therapy, as discussed below. A host cell containing nucleic acidaccording to the present invention, e.g. as a result of introduction ofthe nucleic acid into the cell or into an ancestor of the cell and/orgenetic alteration of the sequence endogenous to the cell or ancestor(which introduction or alteration may take place in vivo or ex vivo),may be comprised (e.g. in the soma) within an organism which is ananimal, particularly a mammal, which may be human or non-human, such asrabbit, guinea pig, rat, mouse or other rodent, cat, dog, pig, sheep,goat, cattle or horse, or which is a bird, such as a chicken.Genetically modified or transgenic animals or birds comprising such acell are also provided as further aspects of the present invention.

[0172] In another aspect of the invention, there is provided a methodfor producing a transgenic non-human mammal, particularly a rodent suchas a mouse, by incorporating a lesion into the locus of a STRAP gene.

[0173] This may be achieved in a variety of ways. A typical strategy isto use targeted homologous recombination to replace, modify or deletethe wild-type STRAP gene in an embryonic stem (ES) cell. An targetingvector is introduced into ES cells by electroporation, lipofection ormicroinjection. In a few ES cells, the targeting vector pairs with thecognate chromosomal DNA sequence and transfers the desired mutationcarried by the vector into the genome by homologous recombination.Screening or enrichment procedures are used to identify the transfectedcells, and a transfected cell is cloned and maintained as a purepopulation. Next, the altered ES cells are injected into the blastocystof a preimplantation mouse embryo or alternatively an aggregationchimera is prepared in which the ES cells are placed between twoblastocysts which, with the ES cells, merge to form a single chimericblastocyst. The chimeric blastocyst is surgically transferred into theuterus of a foster mother where the development is allowed to progressto term. The resulting animal will be a chimera of normal and donorcells. Typically the donor cells will be from an animal with a clearlydistinguishable phenotype such as skin colour, so that the chimericprogeny is easily identified. The progeny is then bred and itsdescendants cross-bred, giving rise to heterozygotes and homozygotes forthe targeted mutation. The production of transgenic animals is describedfurther by Capecchi, M, R., 1989, Science 244; 1288-1292; Valancius andSmithies, 1991, Mol. Cell. Biol. 11; 1402-1408; and Hasty et al, 1991,Nature 350; 243-246, the disclosures of which are incorporated herein byreference.

[0174] Homologous recombination in gene targeting may be used to replacethe wild-type STRAP gene with a specifically defined mutant form (e.gtruncated or containing one or more substitutions).

[0175] The invention may also be used to replace the wild-type gene witha modified gene capable of expressing a wild-type or otherwise activeSTRAP polypeptide, where the expression may be selectively blockedeither permanently or temporarily. Permanent blocking may be achieved bysupplying means to delete the gene in response to a signal. An exampleof such a means is the cre-lox system where phage lox sites are providedat either end of the transgene, or at least between a sufficient portionthereof (e.g. in two exons located either side or one or more introns).Expression of a cre recombinase causes excision and circularisation ofthe nuclei acid between the two lox sites. Various lines of transgenicanimals, particularly mice, are currently available in the art whichexpress cre recombinase in a developmentally or tissue restrictedmanner, see for example Tsien, Cell, Vol.87(7): 1317-1326, (1996) andBetz, Current Biology, Vol.6(10): 1307-1316 (1996). These animals may becrossed with lox transgenic animals of the invention to examine thefunction of the STRAP gene. An alternative mechanism of control is tosupply a promoter from a tetracyline resistance gene, tet, to thecontrol regions of the STRAP locus such that addition of tetracyline toa cell binds to the promoter and blocks expression of the STRAP gene.

[0176] Transgenic targeting techniques may also be used to delete theSTRAP gene. Methods of targeted gene deletion are described by Brenneret al, WO94/21787 (Cell Genesys), the disclosure of which isincorporated herein by reference.

[0177] Homologous recombination may also be used to produce “knock in”animals which express a polypeptide of the invention in the form of afusion protein, fused to a detectable tag such as β-galactosidase orgreen fluorescent protein. Such transgenic non-human mammals may be usedin methods of determining temporal and spatial expression of the STRAPgene by monitoring the expression of the detectable tag.

[0178] A further alternative is to target control sequences responsiblefor expression of the STRAP gene.

[0179] The invention extends to transgenic non-human mammals obtainableby such methods and to their progeny. Such mammals may be homozygous orheterozygous. Such mammals include mice, rodents, rabbits, sheep, goats,pigs.

[0180] Transgenic non-human mammals may be used for experimentalpurposes in studying the role of STRAP in regulating the cell cycle andin the development of therapies designed to target the interaction ofSTRAP with other cellular factors, particularly p300 and JMY. By“experimental” it is meant permissible for use in animal experimentationor testing purposes under prevailing legislation applicable to theresearch facility where such experimentation occurs.

[0181] Instead of or as well as being used for the production of apolypeptide encoded by a transgene, host cells may be used as a nucleicacid factory to replicate the nucleic acid of interest in order togenerate large amounts of it. Multiple copies of nucleic acid ofinterest may be made within a cell when coupled to an amplifiable genesuch as dihyrofolate reductase (DHFR), as is well known. Host cellstransformed with nucleic acid of interest, or which are descended fromhost cells into which nucleic acid was introduced, may be cultured undersuitable conditions, e.g. in a fermentor, taken from the culture andsubjected to processing to purify the nucleic acid. Followingpurification, the nucleic acid or one or more fragments thereof may beused as desired.

[0182] The provision of the novel STRAP polypeptide also enables, forthe first time, the production of antibodies able to bind specificallyto this molecule.

[0183] Accordingly, a further aspect of the present invention providesan antibody able to bind specifically to the polypeptide whose sequenceis given in a figure herein. Such an antibody may be specific in thesense of being able to distinguish between the polypeptide it is able tobind and other human polypeptides for which it has no or substantiallyno binding affinity (e.g. a binding affinity of about 1000× less).Specific antibodies bind an epitope on the molecule which is either notpresent or is not accessible on other molecules. Antibodies according tothe present invention may be specific for the wild-type polypeptide.Antibodies according to the invention may be specific for a particularmutant, variant, allele or derivative polypeptide as between thatmolecule and the wild-type polypeptide, so as to be useful in diagnosticand prognostic methods as discussed below. Antibodies are also useful inpurifying the polypeptide or polypeptides to which they bind, e.g.following production by recombinant expression from encoding nucleicacid.

[0184] Preferred antibodies according to the invention are isolated, inthe sense of being free from contaminants such as antibodies able tobind other polypeptides and/or free of serum components. Monoclonalantibodies are preferred for some purposes, though polyclonal antibodiesare within the scope of the present invention.

[0185] The invention further provides immunological assays whichcomprise:

[0186] (a) bringing a body sample from said subject into contact, underbinding conditions, with an antibody of the invention; and

[0187] (b) determining whether said antibody has been able to bind to apolypeptide in said sample.

[0188] Antibodies may be obtained using techniques which are standard inthe art. Methods of producing antibodies include immunising a mammal(e.g., mouse, rat, rabbit, horse, goat, sheep or monkey) with theprotein or a fragment thereof. Antibodies may be obtained from immunisedanimals using any of a variety of techniques known in the art, andscreened, preferably using binding of antibody to antigen of interest.For instance, Western blotting techniques or immunoprecipitation may beused (Armitage et al., 1992, Nature 357: 80-82). Isolation of antibodiesand/or antibody-producing cells from an animal may be accompanied by astep of sacrificing the animal.

[0189] As an alternative or supplement to immunising a mammal with apeptide, an antibody specific for a protein may be obtained from arecombinantly produced library of expressed immunoglobulin variabledomains, e.g. using lambda bacteriophage or filamentous bacteriophagewhich display functional immunoglobulin binding domains on theirsurfaces; for instance see WO92/01047.

[0190] Suitable peptides for use in immunising an animal and/orisolating anti-STRAP antibody include peptides which have the residues36-46 of FIG. 1 (SEQ ID NO:1 or SEQ ID NO:2).

[0191] Antibodies according to the present invention may be modified ina number of ways. Indeed, the term “antibody” should be construed ascovering antibody fragments such as Fab and scFv fragments, derivatives,functional equivalents and homologues of antibodies, including syntheticmolecules and molecules whose shape mimicks that of an antibody enablingit to bind an antigen or epitope.

[0192] The reactivities of antibodies on a sample may be determined byany appropriate means. Tagging with individual reporter molecules is onepossibility. The reporter molecules may directly or indirectly generatedetectable, and preferably measurable, signals. The linkage of reportermolecules may be directly or indirectly, covalently, e.g. via a peptidebond or non-covalently. Linkage via a peptide bond may be as a result ofrecombinant expression of a gene fusion encoding antibody and reportermolecule.

[0193] One favoured mode is by covalent linkage of each antibody with anindividual fluorochrome, phosphor or laser dye with spectrally isolatedabsorption or emission characteristics. Suitable fluorochromes includefluorescein, rhodamine, phycoerythrin and Texas Red. Suitablechromogenic dyes include diaminobenzidine.

[0194] Other reporters include macromolecular colloidal particles orparticulate material such as latex beads that are coloured, magnetic orparamagnetic, and biologically or chemically active agents that candirectly or indirectly cause detectable signals to be visually observed,electronically detected or otherwise recorded. These molecules may beenzymes which catalyse reactions that develop or change colours or causechanges in electrical properties, for example. They may be molecularlyexcitable, such that electronic transitions between energy states resultin characteristic spectral absorptions or emissions. They may includechemical entities used in conjunction with biosensors. Biotin/avidin orbiotin/streptavidin and alkaline phosphatase detection systems may beemployed.

[0195] Antibodies according to the present invention may be used inscreening for the presence of a polypeptide, for example in a testsample containing cells or cell lysate as discussed, and may be used inpurifying and/or isolating a polypeptide according to the presentinvention, for instance following production of the polypeptide byexpression from encoding nucleic acid therefor. Antibodies may modulatethe activity of the polypeptide to which they bind and so may be usefulin a therapeutic context (which may include prophylaxis) to modulate thep53 dependent response to cellular stress.

[0196] The present invention also provides a substance as describedherein for use in a pharmaceutical composition for the modulation of thep53 dependent cell stress response in an individual. Whether it is apolypeptide, antibody, peptide, nucleic acid molecule, small molecule orother pharmaceutically useful compound according to the presentinvention that is to be given to an individual, administration ispreferably in a “prophylactically effective amount” or a“therapeutically effective amount” (as the case may be, althoughprophylaxis may be considered therapy), this being sufficient to showbenefit to the individual. The actual amount administered, and rate andtime-course of administration, will depend on the nature and severity ofwhat is being treated. Prescription of treatment, e.g. decisions ondosage etc, is within the responsibility of general practitioners andother medical doctors.

[0197] A composition may be administered alone or in combination withother treatments, either simultaneously or sequentially dependent uponthe condition to be treated.

[0198] Pharmaceutical compositions according to the present invention,and for use in accordance with the present invention, may include, inaddition to active ingredient, a pharmaceutically acceptable excipient,carrier, buffer, stabiliser or other materials well known to thoseskilled in the art. Such materials should be non-toxic and should notinterfere with the efficacy of the active ingredient. The precise natureof the carrier or other material will depend on the route ofadministration, which may be oral, or by injection, e.g. cutaneous,subcutaneous or intravenous.

[0199] Pharmaceutical compositions for oral administration may be intablet, capsule, powder or liquid form. A tablet may include a solidcarrier such as gelatin or an adjuvant. Liquid pharmaceuticalcompositions generally include a liquid carrier such as water,petroleum, animal or vegetable oils, mineral oil or synthetic oil.Physiological saline solution, dextrose or other saccharide solution orglycols such as ethylene glycol, propylene glycol or polyethylene glycolmay be included.

[0200] For intravenous, cutaneous or subcutaneous injection, orinjection at the site of affliction, the active ingredient will be inthe form of a parenterally acceptable aqueous solution which ispyrogen-free and has suitable pH, isotonicity and stability. Those ofrelevant skill in the art are well able to prepare suitable solutionsusing, for example, isotonic vehicles such as Sodium Chloride Injection,Ringer's Injection, or Lactated Ringer's Injection. Preservatives,stabilisers, buffers, antioxidants and/or other additives may beincluded, as required.

[0201] Targeting therapies may be used to deliver the active agent morespecifically to certain types of cell, by the use of targeting systemssuch as antibody or cell specific ligands. Targeting may be desirablefor a variety of reasons; for example if the agent is unacceptablytoxic, or if it would otherwise require too high a dosage, or if itwould not otherwise be able to enter the target cells.

[0202] Instead of administering an agent directly, it may be produced intarget cells by expression from an encoding gene introduced into thecells, e.g. in a viral vector (see below). The vector may be targeted tothe specific cells to be treated, or it may contain regulatory elementswhich are switched on more or less selectively by the target cells.Viral vectors may be targeted using specific binding molecules, such asa sugar, glycolipid or protein such as an antibody or binding fragmentthereof. Nucleic acid may be targeted by means of linkage to a proteinligand (such as an antibody or binding fragment thereof) via polylysine,with the ligand being specific for a receptor present on the surface ofthe target cells.

[0203] An agent may be administered in a precursor form, for conversionto an active form by an activating agent produced in, or targeted to,the cells to be treated. Pharmaceutical compositions as described hereinmay be used for anti-sense regulation of gene expression, e.g. targetingan antisense nucleic acid molecule to cells in which a mutant form ofthe gene is expressed, the aim being to reduce production of the mutantgene product. Other approaches to specific down-regulation of genes arewell known, including the use of ribozymes designed to cleave specificnucleic acid sequences. Ribozymes are nucleic acid molecules, actuallyRNA, which specifically cleave single-stranded RNA, such as mRNA, atdefined sequences, and their specificity can be engineered. Hammerheadribozymes may be preferred because they recognise base sequences ofabout 11-18 bases in length, and so have greater specificity thanribozymes of the Tetrahymena type which recognise sequences of about 4bases in length, though the latter type of ribozymes are useful incertain circumstances. References on the use of ribozymes includeMarschall, et al. Cellular and Molecular Neurobiology, 1994. 14(5): 523;Hasselhoff, Nature 334: 585 (1988) and Cech, J. Amer. Med. Assn., 260:3030 (1988).

[0204] Aspects of the present invention will now be illustrated withreference to the accompanying figures described below by experimentalexemplification, by way of example and not limitation. Further aspectsand embodiments will be apparent to those of ordinary skill in the art.All documents mentioned in this specification are hereby incorporatedherein by reference.

EXPERIMENTAL

[0205] Materials and Methods

[0206] Plasmids

[0207] The following plasmids were used; pCMV-p300, pCMV-JMY, pVP16-JMY,pG4-p300, pET-JMY, pCMV-HA-JMY469-558, pCMV-HA-JMY1-403,PCMV-HA-JMY119-403, pET-JMY1-504, PCMV-HA-JMY502-983, pCMV-HA-JMY1-119,pCMV-HA-JMY468-558, pCMV-HA-JMY683-983 and pCMV-HA-NAP2 were asdescribed (Shikama et al., 2000; submitted) Flag-tagged p300,pGEXT-2T-p300744-1571, pGEXT-2T-p3001572-2370, pCMV-p300619-1303,pCMV-p3001257-2284, pCMV-p3001257-19 21, pCMV-p53, pCMV-p5322/23, (Linet al., 1994), pBax-luc (Miyashita and Reed, 1995), pWWP-luc (El-Deiryet al., 1993), pCMV β-gal (Zamanian and La Thangue, 1992), pG5-luc andpTG13 (Lee et al., 1998) and pCMV-HDM2 (Loughran and La Thangue, 2000).

[0208] For the preparation of the STRAP expression vectors,pG4-STRAP8-438 was made by subcloning the appropriate region of theSTRAP cDNA into EcOR1/NotI sites. pVP16-STRAP8-123 was made bysubcloning into BamHI/XhoI site. pETSTRAP8-438 and pETSTRAP8-123 wereconstructed by subcloning into pET28A and pET28C respectively, digestedwith BamHI/XhoI. pCMV-HA-STRAP8-438, pCMV-HA-STRAP8-205,pCMV-HA-STRAP123-205, PCMV-HA-STRAP206-438, pCMV-HA-STRAP206-327 andpCMV-HA-STRAP328-438 were created by subcloning the appropriate cDNAfragment into the BamHI/XhoI site or for pCMV-HA-STRAP8-123 into theXhoI/XbaI site of pCMV-HA vector.

[0209] Antisera.

[0210] The following antisera were used; the p53 monoclonal antibodyDO-1 (Santa Cruz), anti-HA monoclonal antibody Y-11 (BoehringerMannheim), anti-p300 monoclonal antibody Ab-1 (Calbiochem), anti-p300rabbit polyclonal C20 (Santa Cruz), anti-p300 rabbit polyclonal antibodyN-15 (Santa Cruz) and anti-JMY rabbit polyclonal antibody 789 (Shikamaet al., 1999). The STRAP rabbit anti-peptide antisera, 15, was preparedby standard procedures against a STRAP peptide representing from residue30 to 46.

[0211] Isolation of STRAP.

[0212] The yeast strain CTY10.5 containing the LexAβ-galactosidasereporter vector pLex (His) was as previously described (Buck et al.,1995). pLex-JMY was made by inserting into the EcOR1 site in frame thefragment of JMY corresponding to amino acids 469-558 with the DNAbinding domain of LexA. Screening a 10.5 day mouse embryo random primedcDNA library fused to the VP16 trans-activation domain (Vojtek et al.,1993) yielded a single positive clone containing 341 bp of the STRAPcDNA sequence.

[0213] Full-length STRAP cDNAs were isolated through the combinedapproach of screening cDNA libraries prepared from PCC4 mouseteratocarcinoma cells (Stratagene) and RACE (Clontech) using a testiscDNA library.

[0214] Transient Transfections and Reporter Assays.

[0215] For transfection into SAOS2 or U2OS cells, cells were incubatedin DMEM containing 10% serum throughout and transfected with pBax-luc(Miyashita and Reed, 1995), pWWP-luc (El-Deiry et al., 1993), or pTG13(Lee et al., 1998) expression vector for p53 (pCMV-p53), together withthe indicated amounts of the STRAP expression vector and harvested 34-36h post-transfection (Sorensen et al., 1996; Shikama et al., 1999). Alltransfections were performed using the calcium phosphate procedure andincluded the internal control pCMV-β-gal (Zamanian and La Thangue,1992). The mammalian two-hybrid assay was performed in U2OS cells asdescribed using the Gal4-responsive reporter pG5-luc andpG4-p300611-2283 (Lee et al., 1998) together with pVP16-STRAP, orpG4-STRAP hybrid together with pVP16-JMY.

[0216] Immunoprecipitation and Immunoblotting.

[0217] For immunoprecipitation, p53−/− MEFs, U2OS and SAOS2 cells weretransfected with the expression vectors pCMV-p300 and pCMV-JMY andpCMV-HA-STRAP. After 48 h, cells were harvested in TNN buffer (50 mMTris-HCl (pH7.4), 5 mM EDTA, 0.5% NP40, 50 mM NaF, 1 mM DTT, 0.2 mMsodium orthovanadate) and protease inhibitor cocktail (1 mM PMSF,leupeptin, aprotinin and pepstatin (1 μg/ml)) containing 120 mM NaCl andincubated on ice for 30 min.

[0218] The cell extract was first precleared for 1 h with protein G andthen immunoprecipitated with the anti-HA monoclonal antibody Y11, whichwas collected with protein-A agarose. The agarose beads were collectedand washed three times in the extraction buffer before denaturation andSDS-PAGE. Immunoblotting was subsequently performed with either ananti-p300 monoclonal antibody Ab-1 or anti-JMY polyclonal antibody.

[0219] For p53 stability studies p53−/− MEF, were transfected asdescribed with pCMV-p53, pCMV-HDM2 and the indicated amounts ofpCMV-HA-STRAP. 6 h after transfection the cells were harvested asdescribed and submitted to SDS-PAGE and immunoblot. In the case of U2OScells, endogenous p53 protein was followed after the transfection ofpCMV-HA-STRAP. Cells were harvested and the cell extracts prepared asdescribed above. The p53 protein was detected by immunoblotting withDO-1.

[0220] Biochemical Binding Assay.

[0221] Flag-tagged full-length p300 protein was expressed in thebaculovirus expression system and bound to M2-anti-flag antibody agarose(Kodak). For the control beads, the M2-anti-flag antibody agarose wastreated with cell extract from uninfected Sf9 cells.

[0222] The various JMY and STRAP proteins were in vitro translated byusing TNT-coupled reticulocyte lysate system (Promega) in the presenceof [35S]Met/Cys (Amersham). Each translation reaction (25 μl) wasincubated either with the beads coupled to the flag-tagged p300 or thecontrol beads for 2 h at 4° C. The protein complex on the beads waswashed three times in TNN buffer containing 100 mM NaCl and eluted in2×SDS sample buffer and loaded on to an SDS-PAGE gel. The proteins weredetected by autoradiography.

[0223] For JMY469-558, in vitro translation was carried out in theabsence of radioactive amino acids. The protein was detected byimmunoblotting using anti-HA Y11 monoclonal antibody. Wild-type STRAP inpET28a (Novagen) was expressed and purified through His-tagchromatography according to the manufacturer's instructions (Pharmacia).

[0224] The eluted fraction containing His-STRAP was dialysed against 50mM Tris (pH 7.5), 100 mM KCl, 20% glycerol, 0.2 mM DTT, 0.2 mM PMSF. Forthe in vitro binding assay, flag-tagged full-length p300 was expressedin the baculovirus system and bound to M2-anti-flag antibody agarose(Kodak). For control beads, the M2-anti-flag antibody agarose wastreated with cell extract from uninfected Sf9 cells. Wild-type JMY andSTRAP in pET28a vector (Novagen) were expressed in bacteria and purifiedthrough nickel chromatography according to the manufacturer'sinstructions (Pharmacia). The eluted fraction containing His-STRAP andHis-JMY protein was dialysed against 50 mM Tris (pH 7.5), 100 mM KCl,20% glycerol, 0.2 mM DTT, 0.2 mM PMSF.

[0225] The binding assay using flag-p300 was performed for 3 h at 4° C.in a buffer containing Tris-pH 7.5, 250 mM NaCl, 0.1% NP40, 10%glycerol, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.5 mg/ml BSA, 0.5 mM DTT, 0.5 mMPMSF and protease inhibitors. After binding, the beads were washed threetimes in 50 mM Tris-pH 7.5, 250 mM NaCl, 0.1% NP40, 10% glycerol, 1.5 mMMgCl2, 0.2 mM EDTA, 0.1 mM DTT, 0.1 mM PMSF and protease inhibitors. Thelevel of JMY bound to p300 was detected by immunoblotting with theanti-JMY antiserum.

[0226] Cyclohexamide Treatment.

[0227] U2OS cells were transfected with the pCMV-HA-STRAP, pCMV-HA-JMY,pCMV-p300 and for control empty pcDNA3 expression vectors. After 48 h oftransfection, cyclohexamide (10 μg/ml) was added to the cells for theindicated time periods and cells were harvested in TNN buffer (Maki andHowley, 1997). The cell extract was loaded on SDS-PAGE and the p53protein was detected on nitrocellulose membrane with anti-p53 monoclonalantibody DO1.

[0228] Etoposide Treatment

[0229] Cells were transfected with the pCMV-HA-STRAP, pCMV-JMY orpCMV-p53 and for the control empty pcDNA3 expression vector. Etoposideat a final concentration of 200 nM and 400 nM was added to the cells 12h before harvesting. Cells were harvested in TNN buffer and submitted toeither immunoblotting or luciferase activity assays.

[0230] Flow Cytometry.

[0231] SAOS2 cells were transfected with pCMV-p53 or pCMV-p5322/23 (5□g) either alone or together with pCMV-STPAP. Flow cytometry wasperformed as described previously (de la Luna et al. 1999; Shikama etal., 1999).

[0232] Irradiation.

[0233] Two hours after irradiation (10GyIR and 80J/m2 UV) A31, p53−/−and p53−/−/MDM2−/− MEFs, were washed twice with ice cold PBS (pH7.4).Lysis was performed on ice for 20 min in lysis buffer TNN (Tris HClpH7.5, 50 mM, NaCl 120 mM, EDTA 1 mM, 0.5% Nonidet P-40, 1 mM PMSF,protease inhibitors). Lysates were centrifuged for 20 min, 12,000 rpm,at 4° C. and equal amounts of protein were loaded onto an SDS gel andthe amount of STRAP1 determined after immunoblotting with the anti-STRAPantibody.

[0234] Results

[0235] Isolation and Characterization of STRAP.

[0236] A yeast two-hybrid assay was used to screen for proteins that arecapable of physically interacting with the JMY co-factor, which is knownto form a complex with p300 (Shikama et al., 1999). For the bait, weused a hybrid protein in which an internal domain of JMY from residue469 to 558, containing a region previously assigned to the interactionwith p300 (Shikama et al., 1999), was fused to the LexA DNA bindingdomain.

[0237] Screening of a 10.5 d.p.c. mouse embryo activation domain-taggedlibrary identified a partial cDNA clone of novel sequence. Subsequentisolation and sequence analysis of the full-length cDNA indicated thatit encoded a protein of 440 residues which lacks significant homology toany other known protein or nucleotide sequence on the currentlyavailable data bases (FIG. 1). Because of the properties of this newprotein in performing a key role in facilitating stress-responsiveprotein-protein interactions within the p300 co-activator complex, theprotein has been designated STRAP, to reflect its function as astress-responsive activator of p300.

[0238] A remarkable feature of STRAP is the presence of sixtetratricopeptide repeat (TPR) motifs that are distributed throughoutthe entire length of the protein (FIGS. 4 and 5). Whilst a variety ofother proteins have been found to possess TPR motifs (Lamb et al., 1995;Blatch and Lassle, 1999), STRAP appears to be quite unusual in itstandem distribution of TPR motifs. Based on multiple sequencealignments, there appear to be no strictly conserved residues in the 34amino acid residue TPR motif (Blatch and Lassle, 1999). There arehowever, strong preferences for small hydrophobic residues at certainpositions. The TPR motifs in STRAP fit in well with the existinginformation on the composition of TPR motifs.

[0239] An analysis of the pattern of expression of STRAP by northernblotting with RNA prepared from different mouse tissues including heart,brain, spleen, lung, liver, kidney and testis, indicated that expressionis widespread, similar results being obtained in cell lines derived fromdiverse origins.

[0240] STRAP interacts with distinct components of the p300 co-activatorcomplex.

[0241] The TPR motif is a helical motif that can function inprotein-protein interactions (Lamb et al., 1995; Blatch and Lassle,1999). Since STRAP was isolated in a yeast two-hybrid screen using adomain of JMY as the bait, we assessed if STRAP could bind to JMY inmammalian cells and thereafter studied its interaction with othercomponents of the co-activator complex.

[0242] We performed a series of two-hybrid assays in U2OS cells, inwhich we found that VP16-JMY could induce the activity of a hybridprotein in which the STRAP sequence was fused to the Gal4 DNA bindingdomain, referred to as G4-STRAP, but not G4 alone (FIG. 5). Taking asimilar approach, VP16-STRAP could induce G4-p300 (FIG. 6). Theseresults provide indication that STRAP interacts with at least twocomponents of the p300 co-activator complex, namely JMY and p300.

[0243] U2OS cells were transfected with expression vectors for HA-taggedSTRAP together with JMY or p300, and assessed their interaction byimmunoprecipitation with an anti-HA monoclonal antibody followed byimmunoblotting with either anti-JMY or anti-p300. In both experiments,STRAP specifically co-immunoprecipitated with JMY and p300.

[0244] An anti-peptide antibody against STRAP was prepared to determinewhether similar interactions occur under physiological conditions. Thisantibody reacted specifically with bacterially expressed wild-typeSTRAP, and recognised the exogenous STRAP polypeptide, of about 60,000molecular weight, in transfected cells. Moreover, an endogenouspolypeptide of similar molecular weight was recognised by the anti-STRAPantibody in murine A31 cells. Binding of the antibody to the 60,000molecular weight STRAP polypeptide was blocked upon the presence of thehomologous peptide. We used this anti-STRAP antibody to test whetherp300 and STRAP form a complex under physiological conditions. Inanti-p300 immunoprecipitates from murine A31 cells, we found STRAP to bepresent in the p300 immunocomplex. These results provide strongindication that STRAP and p300 exist as a complex under normalphysiological conditions.

[0245] Binding domains in STRAP, p300 and JMY.

[0246] The binding domains in STRAP for JMY and p300 were determinedthrough a biochemical assay in which different regions of STRAP were invitro translated and thereafter assessed for binding to eitherhis-tagged wild-type JMY or flag-tagged wild-type p300.

[0247] Out of a panel of STRAP derivatives, two distinct regions werefound to be responsible for the interaction with JMY, one located in theN-terminal region up to residue 123, and the other within residue 123 to205; the C-terminal half of STRAP (from residue 206) exhibited littlebinding activity for JMY.

[0248] In a continuation of this analysis, we studied the interactiondomain in STRAP for p300. An analysis of the same set of STRAP mutantderivatives indicated that the predominant p300 interaction domain was,in contrast, localised in the C-terminal half of STRAP, and encompassedfrom residue 206 to 438. Overall, these binding studies established thatSTRAP possesses two separable and distinct regions of interaction forJMY and p300, the binding domain for JMY being primarily localisedwithin the N-terminal half with the p300 interaction domain beingpresent in the C-terminal half (FIG. 7).

[0249] A similar approach was taken to elucidate the domains in JMY andp300 that are responsible for interacting with STRAP. An analysis of thebinding properties of a panel of in vitro translated JMY derivativesindicated that JMY harbours at least two interaction domains for STRAP,one of which resides within the N-terminal 119 residues, the other beingbroadly defined to the C-terminal region from residue 683 to 983. Inaddition, another STRAP binding domain in JMY mapped to residue 468 to558, as this region was used in the two-hybrid screen to isolate STRAP.Thus, JMY contains at least three distinct interaction domains for STRAP(FIG. 7).

[0250] Finally, we investigated a panel of p300 derivatives for theirSTRAP binding activity. As expected, wild-type p300 bound to STRAP, andfurther analysis of the binding properties of the mutant derivativesmapped two p300 interaction domains, one to the N-terminal 595 residues,with the other one located in the C-terminal region between residues1572 to 1921.

[0251] Taken together, this analysis of the binding properties of STRAP,JMY and p300 established that STRAP can bind specifically to JMY andp300, and that it does so through distinct domains within the N- andC-terminal regions of the protein (see FIG. 7). Similarly, JMY and p300possess dedicated domains that allow each protein to interact with STRAPand, as previously documented, with each other.

[0252] STRAP facilitates the interaction between JMY and p300.

[0253] The level of JMY that co-immunoprecipitated with p300 from cellstransfected with JMY and p300 was determined, and thereafter the effectof co-expressing STRAP. At the same time, we investigated the level ofJMY, p300 and STRAP in transfected cells which indicated thatco-expressing STRAP, p300 and JMY caused an accumulation of JMY andSTRAP. In the same cell extract we found, as expected (Shikama et al.,1999), that JMY co-immunoprecipitated with p300 and, importantly, thatthere was a highly significant increase in the level of JMY in the p300immunocomplex in the presence of STRAP. Moreover, since the amount ofp300 in the immunocomplex was similar in the absence or presence ofSTRAP, the increased level of co-immunoprecipitated JMY results from theinfluence of STRAP on the recruitment of JMY into the p300 complex.

[0254] A mammalian two-hybrid assay was used to investigate whetherSTRAP affected the interaction between JMY and p300. As in previousstudies, in a two-hybrid assay G4-p300 and VP16-JMY could interact witheach other (Shikama et al., 1999). Under these conditions, STRAP causeda significant increase in activity. A titration of STRAP indicated thatfurther increases in the amount of co-transfected STRAP caused areduction in the two-hybrid signal (FIG. 8). A possible explanation forthis phenomenon is that there is an optimal level of STRAP that favoursthe interaction between JMY and p300, above which the level of STRAPout-titrates the amount of p300 and JMY, and rather than recruiting p300and JMY into a ternary complex, STRAP binds to each as heterodimer andthus interferes with the two-hybrid interaction.

[0255] We then purified recombinant p300, JMY and STRAP and studied theinfluence of STRAP upon the interaction between JMY and p300 in abiochemical assay. Previous studies have established that JMY and p300can form a protein complex, and identified the domains in each proteinthat are responsible for this interaction (Shikama et al., 1999). Underconditions where purified recombinant p300 and JMY could weakly bind toeach other, the addition of STRAP increased the efficiency of theirinteraction, enhancing the amount of JMY that bound to p300. Thespecificity of this effect was established through a variety of controltreatments, including the effect of a STRAP mutant derivative containingthe N-terminal region (residue 8 to 123), that harbours one complete TPRmotif, and is capable of binding to JMY, but not p300 (FIG. 7). Theaddition of a similar amount of STRAP8-123 failed to affect theinteraction between. p300 and JMY, thus establishing the specific effectof STRAP on the JMY/p300 interaction.

[0256] STRAP augments the p53 response.

[0257] It is known that p300/CBP proteins participate in the control ofp53-dependent transcription, and that during the p53 response alteredp53 stability serves as a major level of regulation through a processwhich perhaps involves p300 (Ko and Prives, 1997; Levine et al., 1997;Shikama et al., 1997). We introduced STRAP into U2OS cells and studiedthe level of endogenous p53 by immunoblotting. Increasing the levels ofSTRAP caused a concomitant increase in p53. Similarly, upon theintroduction of exogenous p53 into p53−/− MEFs, we observed asignificant increase in p53 levels. Mutant derivatives of STRAPrepresenting both N-and C-terminal deletions failed to cause anyalteration in p53 level. We introduced STRAP into U2OS cells to test forany effect on the half-life of p53 and studied endogenous p53 aftertreating the cells with cyclohexamide. We found that endogenous p53 hada half-life of about 30 minutes which, upon the expression of STRAP, wassignificantly lengthened to about 240 minutes. In contrast, theexpression of neither JMY nor p300 had a significant impact on p53half-life, which remained at approximately 30 minutes. Thus, STRAPinduces the p53 protein, which it does s6 in part by altering the rateof p53 turnover and increasing p53 half-life.

[0258] Previous studies have established that the physical interactionof MDM2 with the activation domain of p53 abrogates the p53 response bytargeting p53 for degradation (Haupt et al., 1997; Kubbutat et al.,1997). Thus, the level of exogenous p53 in p53−/− MEFs was reduced uponthe co-expression of human MDM2 (referred to as hDM2). Under theseconditions, the presence of STRAP significantly increased the level ofp53, providing indication that STRAP can override the down-regulation ofp53 activity by hDM2, a result that is consistent with the ability ofSTRAP to extend p53 half-life and augment the levels of the p300co-activator complex. It is important to note that under theseexperimental conditions we failed to detect any direct interactionbetween p53 and STRAP.

[0259] As p300/CBP and JMY are involved in regulating p53transcriptional activity, we investigated the effect of STRAP ondifferent p53-responsive promoters. Consistent with its presence in thep300 co-activator complex and the ability to extend p53 half-life, wefound STRAP to be capable of inducing p53 activity on a variety ofdifferent p53-responsive promoters, including waf1, bax and theartificial p53-responsive TG13 promoter (FIG. 9).

[0260] We then investigated whether the ability of STRAP to increase p53protein levels and augment transcription correlated with thephysiological properties of p53, namely the ability to promote cellcycle arrest and apoptosis (Levine, 1997). In SAOS2 (p53−/−) tumourcells we identified conditions where the introduction of wild-type p53caused a low but significant increase in the population of apoptosingcells (FIG. 10). Under these conditions, co-expressing STRAP with p53resulted in a marked increase in the size of the apoptosing cellpopulation, in contrast to the effects of STRAP in the absence of p53,which were minimal. These results establish that STRAP augments theapoptotic activity of p53.

[0261] The presence of STRAP in the p300 co-activator complex providedindication that STRAP can augment p53-dependent apoptosis throughstimulating p53 transcriptional activity. We assessed whether STRAPcould induce p5322/23, which is a mutant p53 derivative that has tworesidues altered in the activation domain, and which is severelycompromised in transcriptional activity (Lin et al., 1994). Incomparison to wild-type p53, p5322/23 was less efficient at inducingapoptosis but still retained a significant level of activity (FIG. 11).However, in contrast to wild-type p53, the apoptotic activity ofp5322/23 was not affected by co-expressing STRAP, a result that isconsistent with a role for STRAP in stimulating p53 transcription.

[0262] Overall, these studies provide indication that STRAP as acomponent of the p300 co-activator complex induces p53 activity in partby increasing the stability of the p53 protein, and further that thiseffect directly influences p53-dependent transcription and therefore thep53 response.

[0263] STRAP is a damage-responsive component of the p300 co-activatorcomplex.

[0264] We investigated whether the protein level was altered in stressedcells by studying the effect of etoposide, which is an agent that canefficiently induce the p53 response (Kaufmann, 1998; Arriola et al.,1999). We compared in U2OS cells the induction of STRAP to p53, as wellas to the induction of JMY and NAP (nucleosomal assembly protein), thelatter serving as a control protein that was un likely to be affected bystress. STRAP protein levels increased in response to etoposide,although not as dramatically as observed for p53; in contrast, JMY andNAP failed to undergo a similar response, with NAP levels declining(FIG. 12).

[0265] We then investigated the p300/JMY complex in etoposide-treatedU2OS cells, in the absence and presence of STRAP. The expression ofSTRAP caused an increase in the level of JMY bound to p300, which showeda further and significant increase upon treating the cells withetoposide. Using the same cell extracts, a marked increase in the levelof co-immunoprecipitated JMY with p300 was observed when STRAPexpressing cells were treated with etoposide (usually about 10-fold incontrast to 2-fold induction of protein level). These results provideindication that STRAP is a stress responsive protein that augments theinteraction between p300 and JMY.

[0266] The role of STRAP in regulating the stress-responsive interactionbetween p300 and JMY was further characterised in a two-hybrid assayusing G4-p300 and VP16-JMY. We found that treating U2OS cells withetoposide (200 nM) caused a marked reduction in the level of two-hybridsignal, and that the expression of STRAP completely restored two-hybridactivity (FIG. 13). This effect required the integrity of the STRAPprotein, as a mutant derivative that failed to interact with p300 butretained the ability to bind to JMY, STRAP8-205, could not overcome theeffect of etoposide upon the two-hybrid interaction between G4-p300 andVP16-JMY We extended these studies to an analysis of the effect of STRAPupon the etoposide-dependent regulation of wild-type p53 transcriptionalactivity. In a similar fashion to the etoposide effect on the p300/JMYinteraction (FIG. 13), at a high concentration of etoposide, a reductionin p53 activity occurred (FIG. 14). Co-expression of STRAP partiallyovercame the etoposide-dependent down-regulation of p53 transcriptionalactivity and furthermore, the effect of STRAP was dependent upon theintegrity of the wild-type protein, since the expression of STRAP8-123interfered with wild-type STRAP activity, most probably-through adominant-negative type of effect (FIG. 14).

[0267] These results provide indication that STRAP plays a direct rolein facilitating the p53 response in conditions of cellular stress. A fargreater association was observed between STRAP and the p53 transcriptioncomplex in etoposide-treated U2OS cells compared to untreated cells,indicating that the association between STRAP and p300, and thereafterp53, is stress-responsive. These results demonstrate that STRAPfunctions in regulating and maintaining p300 co-activator functionduring cellular stress, and that this role of STRAP contributes to p53activity.

[0268] Endogenous STRAP is a stress-responsive protein.

[0269] The stress responsiveness of endogenous STRAP was determinedstress using the anti-STRAP peptide antibody by studying. STRAP levelsin A31 cells after treatment with etoposide, as well as otherstress-inducing agents. STRAP was found to be effectively induced byetoposide whereas the effect of ultra-violet light and ionizingradiation was less marked.

[0270] The importance of p53 and MDM2 in the-stress-regulation ofendogenous STRAP was assessed by monitoring STRAP levels in earlypassage p53−/− or p53−/−/mdm2−/− mouse embryo fibroblasts (MEFs). STRAPwas induced in p53−/− MEFs to a similar extent as that observed inmurine A31 cells. In contrast, little change in STRAP level was observedin the double knockout p53−/−/mdm2−/− cells. These results show thatSTRAP can be induced independently of p53, but indicate that MDM2 caninfluence the stress-responsive induction of STRAP.

[0271] The stress response control of STRAP described herein allows anumber of important conclusions to be made. Firstly, STRAP is astress-responsive protein, since STRAP levels increased in stressedcells. Secondly, the effect and interaction of STRAP with the p53associated p300 complex provide strong indication that STRAP is afunctionally important component of the p53 response. The latterconclusion, in combination with the earlier results on the ability ofSTRAP to foster the interaction between p300 and JMY, show that aprimary function of STRAP is to augment p300 co-activator activity inconditions of the p53 response.

[0272] STRAP is a novel TPR motif protein.

[0273] The characterisation of STRAP has shown that the proteinpossesses a rather unusual domain organisation, since it is composedalmost entirely of a tandem series of TPR motifs (Lamb et al., 1995).The TPR motif has been found in a wide variety of proteins, fromprokaryotes to eukaryotes, and is generally believed to represent anancient protein-protein-interaction module (Blatch and Lassle, 1999).Proteins that contain TPR motifs function in diverse physiologicalprocesses, including the cell cycle, transcription and the stressresponse, where they usually occur as integral components ofmultiprotein complexes.

[0274] The characteristics ascribed to STRAP as a functionally importantcomponent involved in regulating the p300/CBP co-activator complex fitvery well with the generic properties previously assigned to TPRmotif-containing proteins in regulating and mediating the assembly ofmacromolecular protein complexes. Particularly relevant is theconsiderable body of evidence that highlights the multicomponent natureof p300/CBP co-activator complex, in which STRAP plays a role infacilitating the interaction between p300 and JMY (Shikama et al.,1999). Another notable feature is the fact that STRAP has distinct andseparable TPR motif-containing domains that allow it to bind to proteincomponents of the p300 co-activator complex. Moreover, our results implythat this ability of STRAP to interact with p300 and JMY facilitatestheir interaction, suggesting that STRAP may play a role in maintainingthe functional integrity of the p300/CBP co-activator complex.

[0275] STRAP may facilitate the assembly of the p300/CBP co-activatorcomplex.

[0276] It is interesting to speculate on the role of STRAP in thep300/CBP co-activator complex. In this respect, it is noteworthy thatalthough proteins with multiple TPR motifs are found in other proteincomplexes, the TPR motif usually-co-exists with domains dedicated toother functions, such as phosphatase activity in the case of PP5(Ollendorf and Donoghue, 1997). STRAP is unusual in this respect, as themajority of the protein exists as a series of tandem TPR motifs.

[0277] STRAP can interact specifically with JMY and p300, using domainswhich at the biochemical level appear to be non-overlapping. Previousstudies indicated that JMY and p300 can directly and specifically bindto each other (Shikama et al., 1999), an interaction that STRAP enhancesthrough a process that involves the formation of a ternary complexbetween STRAP, p300 and JMY. STRAP may play a role in regulating theassembly of the p300 complex, and represents a key regulatory componentin controlling p300 co-activator activity.

[0278] STRAP is stress-responsive and augments the p53 response. STRAPinduces p53-dependent transcription and facilitates the p53 response(FIG. 15). Given the documented role of the p300 co-activator complex inregulating p53 activity (Gu et al., 1997; Lill et al., 1997; Lee et al.,1998), this activity may result from the ability of STRAP to foster theassembly of the p300 co-activator complex during cellular stress. In itsregulation of p53, an important property was seen in the level of theSTRAP protein, which undergoes a stress-responsive accumulation whencells were treated with agents such as etoposide, which is known toactivate the p53 response by causing double stranded breaks in genomicDNA (Kaufman, 1998). Thus, our study provides indication that there area number of steps through which STRAP influences the p53 response,whereby the initiating event, namely the STRAP facilitated assembly ofthe p300 co-activator complex contributes to a variety of downstreamconsequences, including an extended p53 half-life, in part throughmodulating MDM2 activity, and thereafter the activation of p53 targetgenes that execute the p53 response.

[0279] A new level of control in the p53 response.

[0280] This document describes the first transcription co-factor thathas a dedicated role in regulating the assembly of a transcriptionalco-activator complex and which, furthermore, possesses molecularproperties which allow it to function during cellular stress. Theproperties of STRAP are well-suited to this role. STRAP favours andstrengthens complex formation between p300 and JMY, and undergoesstress-responsive protein accumulation. These properties endow STRAPwith the ability to maintain p300 co-activator activity in adversecellular conditions. The properties of STRAP may have an importantimpact on facilitating the cellular stress response which, in the caseof the p53 response, relies upon the transcriptional activation of a setof targets genes that act as a major driving force in delivering andexecuting the response mechanism.

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1. An isolated polypeptide which includes the amino acid sequence shownin FIG. 1 (SEQ ID NO: 1 or SEQ ID NO: 2).
 2. An isolated polypeptidewhich is an amino acid sequence variant which shows greater than about60% homology with the amino acid sequence shown in FIG. 1 (SEQ ID NO: 1or SEQ ID NO: 2).
 3. A peptide fragment of which binds JMY and/or p300,said peptide fragment being an isolated polyleptide which includes theamino acid sequence shown in FIG. 1 (SEQ ID NO:1 or SEQ ID NO: 2), or anisolated polyeptide which is an amino acid sequence variant which showsgreater than about 60% homology with the amino acid sequence shown inFIG. 1 (SEQ ID NO: 1 or SEQ ID NO: 2).
 4. An isolated nucleic acidmolecule encoding a polypeptide as shown in FIG. 1B (SEQ ID NO:2).
 5. Anexpression vector comprising a nucleic acid according to claim 4operably linked to a regulatory sequence.
 6. A host cell transformedwith an expression vector of claim
 5. 7. A pharmaceutical compositioncomprising a polypeptide or peptide fragment according to any one ofclaims 1 to 3 and a pharmaceutically acceptable excipient or carrier. 8.A method of making a polypeptide according to claim 1 or claim 2comprising culturing a host cell according to claim 6 under conditionsfor expression of said polypeptide.
 9. A method of making a polypeptideaccording to claim 8 comprising testing for binding for JMY or p300. 10.A method according to claim 8 further comprising isolating and/orpurifying said polypeptide.
 11. A method according to claim 10 whereinthe isolated or purified polypeptide is formulated into a compositioncomprising one or more additional components.
 12. An assay method forobtaining an agent able to interact with a polypeptide or fragmentaccording to any one of claims 1 to 3, including: (i) bringing intocontact a said polypeptide or fragment and a putative binding moleculeor other test substance; and (ii) determining interaction or bindingbetween the polypeptide or fragment and the test substance.
 13. An assaymethod for screening for a substance which modulates the binding of apolypeptide according to claim 1 with JMY, including: (i) bringing asaid polypeptide into contact with a JMY polypeptide in the presence ofone or more test substances, (ii) determining the binding of saidpolypeptide to the JMY polypeptide.
 14. An assay method for screeningfor a substance which modulates the binding of a polypeptide accordingto claim 1 with P300, including: (i) bringing a said polypeptide intocontact with a p300 polypeptide in the presence of one or more testsubstances, (ii) determining the binding of said polypeptide to the p300polypeptide.
 15. An assay method for screening for a substance whichmodulates the binding of a polypeptide according to claim 1 with p300and JMY including: (i) bringing a said polypeptide into contact with ap300 polypeptide and a JMY polypeptide in the presence of one or moretest substances, (ii) determining the binding of said polypeptide to thep300 and JMY polypeptides.
 16. An assay method according to any one ofclaims 13 to 15 comprising determining the p53 stress response in thepresence of said test substance.
 17. Use of a polypeptide according toclaim 1 in an assay method for obtaining a substance which modulates thep53 stress response.
 18. An isolated antibody which binds specificallyto a polypeptide according to claim
 1. 19. A substance obtained using anassay method according to any one of claims 13 to
 15. 20. Apharmaceutical composition comprising a substance according to claim 19.21. A method for treatment of a condition related to the p53 stressresponse comprising administration of a composition according to claim20 to an individual.
 22. (Canceled)
 23. A method according to claim 21wherein the condition is selected from cancer therapy-induced toxicity,hyperthermia, hypoxia, stroke, ischemia, acute inflammation, burns orcell aging. 24-25. (Canceled)